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Recruitment of RNA polymerase III to its target promoters

Recruitment of RNA polymerase III to its target promoters Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Recruitment of RNA polymerase III to its target promoters Laura Schramm and Nouria Hernandez Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA A key step in retrieving the information stored in the (bp). This length limit is consistent with the elongation complex genomes of eukaryotes involves the identifica- properties of RNA polymerase III, which recognizes a tion of transcription units and, more specifically, the rec- simple run of T residues as a termination signal. The ognition of promoter sequences by RNA polymerase. In genes transcribed by RNA polymerase III encode RNA eukaryotes, the task of recognizing nuclear gene promot- molecules involved in fundamental metabolic processes, ers and then transcribing the genes is divided among specifically components of the protein synthesis appara- three highly related enzymes, RNA polymerases I, II, and tus and components of the splicing and tRNA processing III. Each of these RNA polymerases is dedicated to the apparatus, as well as RNAs of unknown function. The transcription of specific sets of genes, and each depends RNA polymerase III promoters are more varied in struc- on accessory factors, the so-called transcription factors, ture than the uniform RNA polymerase I promoters, and to recognize its cognate promoter sequences. yet not as diverse as the RNA polymerase II promoters. RNA polymerase I is unique among the nuclear RNA They have been divided into three main types, two of polymerases in transcribing only one set of genes, the which are gene-internal and generally TATA-less, and large, tandemly repeated, ribosomal RNA genes, and one of which is gene-external and contains a TATA box. thus in having to recognize a single promoter structure. Remarkably, we have a good, and in some cases a very RNA polymerase II transcribes the protein-coding genes detailed, understanding of how RNA polymerase III is (mRNA genes) as well as some small nuclear RNA recruited to each of these types of promoters. This pro- (snRNA) genes. The RNA polymerase II promoters can vides a paradigm of how the same enzyme can be re- be divided into a core region, defined as the minimal cruited to different promoter structures through differ- region capable of directing transcription in vitro, and a ent combinations of protein–DNA and protein–protein regulatory region. The regulatory regions are highly var- interactions. Here we summarize our present under- ied in structure, reflecting the highly varied synthesis standing of the various pathways leading to recruitment patterns of cellular proteins and the need for exquisite of RNA polymerase III. Other recent reviews on RNA and complex regulation of these patterns. The core pro- polymerase III transcription include those by Geidus- moters themselves come in different types that, in chek and Kassavetis (2001) and Huang and Maraia (2001). mRNA-encoding genes, can contain a TATA box, an ini- Reviews on the regulation of RNA polymerase III tran- tiator, a downstream promoter element, or various com- scription, which is not covered here, include those by binations thereof. The assembly of a functional RNA Ghavidel et al. (1999) and Brown et al. (2000). polymerase II transcription complex on a promoter con- sisting of just a TATA box has been extensively studied. Structure of RNA polymerase III promoters All the factors involved in the process have been identi- The three types of RNA polymerase III promoters are fied, and much is known about how these factors inter- called types 1–3. The first RNA polymerase III promoters act with DNA and with each other to recruit, eventually, characterized were those of the Xenopus laevis 5S RNA RNA polymerase II (for reviews, see Orphanides et al. gene (Bogenhagen et al. 1980; Sakonju et al. 1980), which 1996; Woychik and Hampsey 2002). How RNA polymer- encodes the small ribosomal RNA, the Adenovirus 2 ase II transcription complexes assemble on TATA-less (Ad2) VAI gene (Fowlkes and Shenk 1980), and various promoters is, however, not as well understood. tRNA genes from X. laevis and Drosophila melanogaster RNA polymerase III is dedicated to the transcription of (Galli et al. 1981; Hofstetter et al. 1981; Sharp et al. an eclectic collection of genes whose main common fea- 1981). The 5S promoter is the only example of a type 1 tures are that they encode structural or catalytic RNAs RNA polymerase III promoter, and the Ad2 VAI and and that they are, as a rule, shorter than 400 base pairs tRNA promoters are typical type 2 promoters. As shown in Figure 1, these promoters are intragenic. The X. laevis Corresponding author. 5S gene promoter consists of an A box, an intermediate E-MAIL [email protected]; FAX (516) 367-6801. element (IE), and a C box that is conserved in the 5S Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1018902. promoters of different species. Together, these elements GENES & DEVELOPMENT 16:2593–2620 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 2593 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez Figure 1. Different types of RNA polymerase III promoters. The type 1 promoter of the Xenopus laevis 5S RNA gene consists of an internal control region (ICR), which can be subdivided into A box (+50 to +60), intermediate element (IE, +67 to +72), and C box (+80 Leu to +90). The type 2 promoter of the X. laevis tRNA gene consists of an A box (+8 to +19) and a B box (+52 to +62). The type 3 promoter of the Homo sapiens U6 snRNA gene consists of a distal sequence element (DSE, −215 to −240) that enhances transcription and a core promoter composed of a proximal sequence element (PSE, −65 to −48) and a TATA box (−32 to −25). The Saccharomyces cerevisiae promoter is a hybrid promoter consisting of a TATA box (−30 to −23), an A box (+21 to +31), and a B box located downstream of the U6 coding region (from +234to +244relative to the start site of transcript ion). constitute the internal control region (ICR; Bogenhagen Clayton 1990), as well as in genes encoding RNAs of 1985; Pieler et al. 1985a,b, 1987). In the Saccharomyces unknown function. Their discovery came as a surprise cerevisiae 5S genes, only the C box is required for tran- because, unlike the then-characterized type 1 and 2 pro- scription (Challice and Segall 1989). moters, the type 3 core promoters turned out to be gene- The Ad2 VAI and most tRNA promoters consist of an external. As illustrated in Figure 1, they are located in A box and a B box (Galli et al. 1981; Hofstetter et al. the 5-flanking region of the gene and consist of a proxi- 1981; Sharp et al. 1981; Allison et al. 1983). These are mal sequence element (PSE), which also constitutes, on well conserved in tRNA genes from various species, its own, the core of RNA polymerase II snRNA promot- probably in part because they encode the tRNA D- and ers, and a TATA box located at a fixed distance down- T-loops, which are required for tRNA function. The stream of the PSE (Hernandez and Lucito 1988; Mattaj et spacing between the A- and B-boxes varies greatly, how- al. 1988; Kunkel and Pederson 1989; Lobo and Hernan- ever, in part to accommodate introns. The A-boxes of dez 1989). Strikingly, in the vertebrate snRNA promot- type 1 and 2 promoters are structurally related and are ers, RNA polymerase specificity can be switched from interchangeable in X. laevis (Ciliberto et al. 1983). How- RNA polymerase III to RNA polymerase II and vice versa ever, this apparently reflects a similarity in sequence by abrogation or generation of the TATA box (Lobo and rather than a conserved function, because, as detailed Hernandez 1989). Upstream of the PSE is an element below, the A-boxes of 5S and tRNA genes bind different referred to as the distal sequence element (DSE), which transcription factors (Braun et al. 1992a). activates transcription from the core promoter. The type 3 core promoters were identified originally in Although the presence of a TATA box is the hallmark mammalian U6 snRNA genes, which encode the U6 of type 3, gene-external, promoters, it is also found in the snRNA component of the spliceosome (Krol et al. 1987; 5-flanking regions of some genes with gene-internal pro- Das et al. 1988; Kunkel and Pederson 1988), and in the moter elements. Figure 1 shows an example of such a human 7SK gene (Murphy et al. 1986), whose RNA prod- hybrid promoter, namely the S. cerevisiae U6 snRNA uct has been recently implicated in the regulation of the promoter. It consists of an A box, a B box located at an CDK9/cyclin T complex (Nguyen et al. 2001; Yang et al. unusual position 120 bp downstream of the RNA coding 2001). They are also found in, for example, the H1 RNA region, and a TATA box located upstream of the tran- gene, which encodes the RNA component of human scription start site. All three of these promoter elements RNase P (Baer et al. 1989), and the gene encoding the are required for efficient transcription in vivo (Brow and RNA component of human RNase MRP (Topper and Guthrie 1990; Eschenlauer et al. 1993). Other examples 2594 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors include some A- and B-box-containing tRNA genes in ered, they were shown to require the B and C fractions, plants (Yukawa et al. 2000), yeast (Dieci et al. 2000), and or the B fraction and a D fraction eluted between 600 silkworm (Ouyang et al. 2000), in which TATA boxes mM and 1000 mM KCl from the phosphocellulose col- present in the 5-flanking region greatly contribute to umn (Lobo et al. 1991). Most of the activities in these transcription efficiency. More recently, an analysis in fractions required for RNA polymerase III transcription Schizosaccharomyces pombe has revealed that in this have now been characterized, both from yeast and hu- organism nearly all tRNA and 5S genes contain a TATA man cells. Figure 2 shows, in a highly simplified manner, box upstream of the transcription start site that is re- how these factors can assemble in an ordered fashion to quired for transcription (Hamada et al. 2001). Strikingly, recruit RNA polymerase III. The green arrows symbolize in vitro, artificial promoters consisting of just a TATA interactions of DNA-binding proteins with promoter el- box can direct RNA polymerase III transcription, indi- ements, the blue arrows protein–protein contacts among cating that under these circumstances, the TATA box various transcription factors, and the purple arrows pro- contains all necessary information to assemble an RNA tein–protein contacts between RNA polymerase III and polymerase III transcription initiation complex (Mitchell transcription factors. et al. 1992; Roberts et al. 1995; Wang and Stumph 1995; In type 2 promoters, the A and B boxes are recognized Whitehall et al. 1995; Huang et al. 1996). by a multisubunit complex in the C fraction called TFIIIC or TFIIIC2 (Lassar et al. 1983). This initial DNA– protein interaction then allows the recruitment of an The assembly pathways directed by the different activity in the B fraction called TFIIIB (Bieker et al. 1985; types of RNA polymerase III promoters converge Setzer and Brown 1985). TFIIIB is composed of three on recruitment of TFIIIB and RNA polymerase III polypeptides, one of which is the TATA-box-binding protein TBP. The binding of TFIIIB to the promoter in The characterization of RNA polymerase III transcrip- turn allows the recruitment of RNA polymerase III, tion factors started with the fractionation of a HeLa cell mainly through protein–protein interactions with extract over a phosphocellulose column into three frac- TFIIIB, although contacts with TFIIIC may also contrib- tions known as fractions A (the phosphocellulose 100 ute (Fig. 2A). In type 1 promoters, the ICR is recognized mM KCl flowthrough), B (a 100 mM–350 mM KCl step by the activity present in the A fraction, a zinc finger elution), and C (a 350 mM–600 mM KCl step elution), protein referred to as TFIIIA (Engelke et al. 1980; Sakonju and the observation that transcription from type 2 pro- moters required fractions B and C, whereas transcription et al. 1981). Formation of the TFIIIA–DNA complex then from type 1 promoters required the three fractions (Se- allows for the binding of TFIIIC (Lassar et al. 1983). gall et al. 1980). After the type 3 promoters were discov- Thus, TFIIIA can be viewed as a specificity factor that Figure 2. Different pathways for recruitment of TFIIIB and RNA polymerase III. The initiation complexes formed on type 2, 1, and 3 promoters, as well as on an artificial promoter consisting of just a TATA box, are shown. The green arrows symbolize interactions of DNA-binding proteins with promoter elements, the blue arrows protein–protein contacts among various transcription factors, and the purple arrows protein–protein contacts between RNA polymerase III and transcription factors. GENES & DEVELOPMENT 2595 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez alters the promoter-recognition properties of TFIIIC and unique system in which we know how different acces- targets it to the 5S promoter. After the binding of TFIIIC, sory factors combine to recruit, ultimately, a TFIIIB ac- the pathway to recruitment of the polymerase is similar tivity and RNA polymerase III. Below, we first describe to that in type 2 promoters, with the recruitment of briefly the subunit composition of RNA polymerase III. TFIIIB and RNA polymerase III (Fig. 2B). In type 3 pro- For a discussion of the likely three-dimensional struc- moters, the PSE is recognized by a multisubunit complex ture of RNA polymerase III, see Geiduschek and Kassa- variously called the PSE-binding protein (PBP), the PSE vetis (2001). We then describe the characterization of its transcription factor (PTF), or the snRNA activating pro- key transcription factor, TFIIIB, both yeast and human, tein complex (SNAP ), and the TATA box is recognized and we summarize our present understanding of how by the TBP component of a specialized TFIIIB-like activ- this factor bridges DNA and RNA polymerase III. We ity (Waldschmidt et al. 1991; Murphy et al. 1992; Sad- then summarize what is known about the various factors owski et al. 1993; Yoon et al. 1995; Schramm et al. 2000; that, in vivo, mediate the recruitment of TFIIIB on most, Teichmann et al. 2000). These DNA–protein interac- if not all, promoters, namely, TFIIIA, TFIIIC, and SNAP . tions are reinforced by protein–protein interactions be- We end with a description of some factors that have been tween SNAP and TBP (Mittal and Hernandez 1997; Ma implicated in termination and recycling of RNA poly- and Hernandez 2002). The binding of SNAP and the merase III. TFIIIB-like activity then lead to recruitment of RNA polymerase III (Sepehri Chong et al. 2001), probably RNA polymerase III through protein–protein contacts with the two DNA- bound factors, SNAP and the TFIIIB-like activity, al- RNA polymerase III is well defined in S. cerevisiae, con- though this has not yet been demonstrated (Fig. 2C). sisting of 17 subunits, as shown in Table 1. All the cor- Figure 2 also shows a recruitment pathway in which responding genes except for RPC37 have been disrupted TFIIIB is directly recruited to a TATA box without the and shown to be essential (for review, see Chedin et al. help of protein–protein contacts with either TFIIIC or 1998). Of the 17 subunits, 10 are unique to RNA poly- SNAP (Fig. 2D). This pathway can be observed in vitro merase III and are designated the C subunits, two are with S. cerevisiae TFIIIB, and, although it is not observed common to RNA polymerases I and III and are desig- in vivo, it reveals a profound aspect of RNA polymerase nated AC subunits, and five are common to the three III transcription, namely, that TFIIIB is sufficient for RNA polymerases and are designated ABC subunits. The RNA polymerase recruitment. TFIIIB was first identified common subunits have different names in RNA poly- as the key RNA polymerase III transcription factor by a merases I and II, as indicated in Table 1 for RNA poly- series of experiments in which S. cerevisiae TFIIIB was merase II. C160, C128, AC40, AC19, and ABC23 are evo- first recruited to either a 5S promoter through prior bind- lutionarily related to the core subunits of Escherichia ing of TFIIIA and TFIIIC, or a tRNA promoter through coli RNA polymerase, as indicated in parentheses in the prior binding of TFIIIC (Kassavetis et al. 1990). TFIIIA table. Of the C subunits, five, indicated in bold in Table and/or TFIIIC were then stripped from the DNA by treat- 1, are specific to RNA polymerase III. ment with heparin or high concentrations of salt. Under Human RNA polymerase III has been purified both by these conditions, functional TFIIIA and TFIIIC were re- conventional chromatography (Wang and Roeder 1996) leased from the templates, but remarkably, TFIIIB re- and from cell lines expressing tagged Homo sapiens (Hs) mained bound to the DNA, generating the same foot- RPC4/RPC53/BN51 (Wang and Roeder 1997), but until print upstream of the transcription start site as it did in recently, only five of its subunits had been characterized: the presence of TFIIIA and/or TFIIIC (Kassavetis et al. HsRPC4/RPC53 (Ittmann et al. 1993; Jackson et al. 1989, 1990). These stripped templates were able to sup- 1995), HsRPC1/RPC155 (Sepehri and Hernandez 1997), port several rounds of properly initiated RNA polymer- HsRPC3/RPC62, HsRPC6/RPC39, and HsRPC7/RPC32 ase III transcription. This suggested that, at least in (Wang and Roeder 1997). Human RNA polymerase III yeast, TFIIIB was sufficient to recruit RNA polymerase has now been purified from a stable cell line expressing III and direct several rounds of transcription, and there- a doubly tagged HsRPC4subunit, and its subunits have fore that the main function of TFIIIA and TFIIIC was to been identified by mass spectrometry (Hu et al. 2002). recruit TFIIIB to the DNA (Kassavetis et al. 1990). With This analysis has resulted in the identification of or- the observation that just a TATA box could direct RNA thologs of all of the yeast RNA polymerase III subunits polymerase III transcription in vitro and with the avail- except for ABC10, which was not detected probably ability of recombinant TFIIIB, it then became possible to because of its small size (7 kD). The newly described confirm that a TATA box could direct several rounds of human subunits were named according to the guide RNA polymerase III transcription with just recombinant shown in the fourth column of Table 1, in which the TFIIIB and highly purified RNA polymerase III (Kassa- yeast C, AC, and ABC subunits were numbered sepa- vetis et al. 1995; Rüth et al. 1996). Thus, TFIIIA, TFIIIC, rately in order of decreasing apparent molecular weight. and SNAP can be viewed as recruitment factors whose Such a nomenclature would provide the same name for main function is to recruit TFIIIB to promoters of various orthologs from different species, as shown in the fourth structures, which then allows the recruitment of RNA and sixth column in Table 1. polymerase III. The characterization of human RPC8 and RPC9 The different RNA polymerase III promoters offer a brought an unexpected result. BLAST searches revealed 2596 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Table 1. Subunits of Saccharomyces cerevisiae and Homo sapiens RNA polymerase III % amino acid Corresponding identities between S. cerevisiae S. cerevisiae H. sapiens and RNA Pol III MW Accession Guide for RNA Pol II H. sapiens RNA MW S. cerevisiae subunits (kD) number nomenclature subunits RNA Pol III subunits (kD) Accession number Pol III subunits C160 (-like) 162.1 P04051 ScRPC1 RPB1 HsRPC1/RPC155 155.6 AAB86536 50% (1356/1391) C128 (-like) 129.3 AAB59324ScRPC2 RPB2 HsRPC2 127.6 AY092084 63%(1115/1133) C82 73.6 CAA45072 ScRPC3 HsRPC3/RPC62 60.5 NP_006459/XP_034604 22% (163/534) C53 46.6 P25441 ScRPC4 HsRPC4/RPC53 44.4 AY092086 28% (134/398) C37 32.1 NP_012950 ScRPC5 HsRPC5 79.8 AY092085 26% (160/708) C34 36.1 P32910 ScRPC6 HsRPC6/RPC39 35.6 NP_006457/XP_009639 26% (216/316) C31 27.7 P17890 ScRPC7 HsRPC7/RPC32 25.9 AAB63676/XP_036456 35% (44/223) C25 24.3 P35718 ScRPC8 RPB7 HsRPC8 22.9 AY092087 42% (201/204) C17 18.6 P47076 ScRPC9 RPB4 HsRPC9/CGRP-RC 16.8 AAC25992 30% (122/148) C11 12.5AAD12060ScRPC10 RPB9 HsRPC10/RPC11 12.3NP_05739452%(108/108 ) AC40 (-like) 37.6 P07703 ScRPAC1 RPB3 HsRPAC1/RPA5,RPA39 38.6 NP_004866 47% (287/342) AC19 (-like) 16.1 P28000 ScRPAC2 RPB11 HsRPAC2/RPA9,RPA16 15.2 NP_057056 45% (119/133) ABC27 25.1 P20434 ScRPABC1 RPB5 HsRPABC1/RPB5,RPB25 24.6 P19388 42% (207/210) ABC23 (-like) 17.9 AAA34989 ScRPABC2 RPB6 HsRPABC2/RPB6,RPB14.4 14.5 P41584 72% (83/127) ABC14.5 16.5 CAA37383 ScRPABC3 RPB8 HsRPABC3/RPB8,RPB17 17.1 P52434 35% (147/150) ABC10 7.7 AAA64417 ScRPABC4 RPB12 HsRPABC4/RPB7.0 7.0 P53803 52% (42/58) ABC10 8.2 P22139 ScRPABC5 RPB10 HsRPABC5/RPB10,RPB7.6 7.6 P52436 73% (67/67) Subunits in bold do not have paralogues in RNA polymerases I and II; those in bold and underlined form a complex separable from the rest of the enzyme. Subunits corresponding to the E. coli , , , and  subunits are indicated. ABC27 and RPB5, ABC23 and RPB6, ABC14.5 and RPB8, ABC10 and RPB12, ABC10 and RPB10 designate in each case the same protein. For HsRPC62, HsRPC39, and HsRPC32, the sequence under the first accession number (Wang and Roeder 1997) differs in several positions from both the sequences deposited by NCBI (second accession number) and genomic sequences. The first number in the parentheses indicates the length of the region of similarity; the second number indicates the total length of the human protein. Reprinted from Hu et al. (2002). that HsRPC8 is related to the RNA polymerase II sub- in transcription initiation, but in this case both subunits unit RPB7, as noted earlier for the S. cerevisiae HsRPC8 are essential for yeast cell viability, perhaps because ortholog C25 (Sadhale and Woychik 1994). In addition, most RNA polymerase III genes encode components es- however, HsRPC9 is related to RPB4, and like RPB4 and sential for cell metabolism. RPB7, which associate with each other and form a dimer The human RNA polymerase III subunits are in gen- detachable from the rest of RNA polymerase II (Edwards eral quite similar to their yeast counterparts with the et al. 1991; Khazak et al. 1998), HsRPC8 and HsRPC9 notable exception of the subunits with no paralogs in associate with each other (Hu et al. 2002). This strongly RNA polymerase II (Jackson et al. 1995; Wang and suggests that HsRPC8 and HsRPC9 are paralogs of RPB7 Roeder 1997; Hu et al. 2002). For example, the human and RPB4, as indicated in Table 1, and that the corre- ortholog of yeast C37 is an 80-kD protein, HsRPC5, sponding S. cerevisiae RNA polymerase III subunits C25 whose similarity to the yeast protein is confined to its and C17 can similarly associate with each other. N-terminal fourth, which shows 26% identity with C37 RPB7, but not RPB4, is essential for yeast cell viability (Hu et al. 2002). Nevertheless, like yeast C37, which (Woychik and Young 1989; McKune et al. 1993). RPB4 associates with the yeast C53 subunit (Flores et al. 1999), is, however, essential for cellular responses to stress HsRPC5 associates with HsRPC4/RPC53, the human or- (Choder and Young 1993) and thus in vivo, the require- tholog of yeast C53, and this association is through the ment for the RPB4subunit may be promoter-specific. HsRPC5 and HsRPC4/RPC53 domains conserved in The RPB4/RPB7 complex is thought to stabilize the open their yeast counterparts (Hu et al. 2002). Interestingly, at promoter complex and perhaps the early transcribing least some of the subunits with no paralogs in RNA poly- complex prior to promoter escape by binding to nascent merase II seem to be involved in promoter recognition. RNA or to single-stranded DNA in the transcription The C82, C34, and C31 subunits (bold and underlined in bubble (Orlicky et al. 2001; Todone et al. 2001). The S. Table 1) dissociate from a yeast enzyme carrying a mu- cerevisiae RNA polymerase III paralogs of RBP7 and tation within the zinc finger domain of the largest sub- RPB4, C25 and C17, are both essential for viability in unit, and each associates with the two others in a yeast yeast (Sadhale and Woychik 1994; Ferri et al. 2000). Two- two-hybrid assay, suggesting that these three subunits hybrid and coimmunoprecipitation experiments indicate form a subcomplex detachable from the rest of the en- that C17 interacts with the transcription initiation fac- zyme (Werner et al. 1992, 1993). In the human enzyme, tor Brf1 and with the RNA polymerase III C31 subunit such a subcomplex could be demonstrated directly by (Ferri et al. 2000), which, as described below, is itself sucrose gradient centrifugation under partially denatur- required for transcription initiation (Werner et al. 1992, ing conditions and by reconstitution of the subcomplex 1993; Wang and Roeder 1997). Thus, the RNA polymer- from recombinant subunits (Wang and Roeder 1997). ase III paralogs of RPB4and RPB7 may also be involved The subunits in the subcomplex are not required for ef- GENES & DEVELOPMENT 2597 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ficient elongation and termination, but are required for ratowski and Zhou 1992). This ability to suppress muta- specific initiation (Thuillier et al. 1995; Brun et al. 1997; tions in TBP suggested that Brf1 might be associated Wang and Roeder 1997). Consistent with this observa- with TBP, and thus that TBP might be part of the TFIIIB tion and as detailed further below, the C34subunit and activity. Indeed, TBP was shown to be part of both the its human counterpart interact directly with TFIIIB sub- yeast and mammalian TFIIIB activity by biochemical units (Werner et al. 1993; Khoo et al. 1994; Wang and methods (Margottin et al. 1991; Huet and Sentenac 1992; Roeder 1997). Kassavetis et al. 1992; Lobo et al. 1992; Taggart et al. 1992; White and Jackson 1992; Chiang et al. 1993; Mey- ers and Sharp 1993), and to constitute a previously un- recognized, Brf1-associated, component of the B activity Composition of TFIIIB (Kassavetis et al. 1992). The cloning of the gene encoding TBP is requiredfor transcription by RNA polymerase yeast B (B, Kassavetis et al. 1995; TFIIIB90, Roberts et III both in S. cerevisiae andhuman cells al. 1996; TFC7p, Rüth et al. 1996), now referred to as Bdp1 (B double prime 1, Willis 2002), then completed the Although the presence of an RNA polymerase III tran- characterization of S. cerevisiae TFIIIB. scription activity in the phosphocellulose B fraction was recognized in the early 1980s, the composition of this activity remained a mystery for the next 10 years. By the Identification of human Brf1 and Brf2 late 1980s, however, the concept that the TATA-box- In S. cerevisiae, all RNA polymerase III promoters re- binding protein TBP was a factor uniquely dedicated to cruit the same TFIIIB factor (Joazeiro et al. 1994). In transcription by RNA polymerase II began to change higher eukaryotes, however, the situation is more com- with the finding that an essential element of the U6 plex, consistent with the need to transcribe much more promoter was an A/T-rich region, that is, a potential complex genomes. Thus, the initial characterization of binding site for TBP. Biochemical fractionation and re- mammalian TFIIIB not only indicated that TBP was part constitution experiments then identified TBP as a factor of the activity (Lobo et al. 1992; Taggart et al. 1992; required for transcription of both the yeast and human White and Jackson 1992), but also that type 1 and 2 pro- U6 snRNA genes (Lobo et al. 1991; Margottin et al. 1991; moters used different components in the TFIIIB fraction Simmen et al. 1991), whose binding to wild-type and than type 3 promoters. Type 1 and 2 promoters were mutant U6 TATA boxes correlated with transcription shown to require a TBP-containing complex (Lobo et al. activity (Lobo et al. 1991). These findings established 1992; Teichmann and Seifart 1995) consisting of TBP and that TATA boxes are part of at least some RNA poly- a homolog of yeast Brf1 (Wang and Roeder 1995; Mital et merase III promoters, and that they act by recruiting al. 1996) referred to as HsBrf1 (Homo sapiens Brf1). TBP. They also raised the possibility that TBP might be Depletion of extracts with antibodies directed against required for RNA polymerase III transcription in general. the C-terminal half of HsBrf1 debilitated transcription Indeed, in vitro competition experiments with TATA- from the type 2 VAI promoter, as expected, but had no containing oligonucleotides then indicated that a TATA- effect on transcription from the type 3 human U6 box-binding factor was required for transcription of the snRNA promoter (Mital et al. 1996; Henry et al. 1998a). VAI and tRNA genes (White et al. 1992), and inactivation On the other hand, depletion of extracts with antibodies of TBP in yeast was shown to lead to defects in transcrip- raised against full-length HsBrf1 or against a peptide de- tion by all three RNA polymerases (Cormack and Struhl rived from the N-terminal portion of the protein inhib- 1992; Schultz et al. 1992). The remaining question was ited transcription from all types of RNA polymerase III how to place TBP in what was then known about RNA promoters, although only transcription from type 1 and 2 polymerase III transcription factors. promoters could be reconstituted by addition of recom- binant HsBrf1 (Wang and Roeder 1995; Schramm et al. 2000). These observations suggested that type 3 promot- Identification of S. cerevisiae Brf1 andBdp1 ers use a protein related to Brf1 in its N-terminal but not Yeast TFIIIB had been shown to consist of two chromato- its C-terminal region, and led to the characterization of a graphically separable activities, named B and B, which new protein, originally called BRFU (Schramm et al. contained polypeptides of 70 and 90 kD, respectively, 2000) or TFIIIB50 (Teichmann et al. 2000), and now re- that could be cross-linked to the DNA (Bartholomew et ferred to as HsBrf2 (Willis 2002). Thus, S. cerevisiae Brf1 al. 1991; Kassavetis et al. 1991). A major step in the com- has at least two homologs in human cells, HsBrf1 and plete characterization of TFIIIB came with the cloning of HsBrf2. the gene encoding the 70-kD polypeptide, now referred Figure 3 shows the structure of TFIIB and various Brf to as Brf1 (TFIIB-related factor 1; for a description of a proteins. H. sapiens and S. cerevisiae (Sc) Brf1 as well as universal nomenclature of TFIIIB components, see Willis HsBrf2 contain, like TFIIB, an N-terminal zinc-binding 2002). The gene was cloned as a suppressor of a tRNA domain (green box) and a “core domain” consisting of gene A-box mutation and called PCF4 (López-De-León et two imperfect repeats (blue box). In addition, the Brf1 al. 1992). It was also cloned, however, as an allele-spe- and Brf2 proteins contain C-terminal domains absent in cific high-copy suppressor of certain mutations in TBP TFIIB. Within the C-terminal segment of Brf1, three re- and called BRF1 (Colbert and Hahn 1992) or TDS4 (Bu- gions, designated regions I, II, and III, are conserved in 2598 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Figure 3. TFIIB, Brf1, and Brf2 form a family of related transcription factors. The location of the structured zinc ribbon as modeled in ScTFIIB and ScBrf1 (Hahn and Roberts 2000) based on the NMR structure of PfTFIIB (Zhu et al. 1996) and that of the corresponding region in HsBrf1 and HsBrf2 is indicated in green. The location of the structured core domain of TFIIB (Bagby et al. 1995; Nikolov et al. 1995) and that of the corresponding regions in the other proteins is indicated in blue. The percentages below the sequences indicate percent identities between HsBrf2 and HsTFIIB, ScTFIIB, ScBrf1, and HsBrf1 within the region of highest conservation (bracketed by the stippled lines) in pairwise alignments performed with BLAST. The purple boxes in the C-terminal regions of ScBrf1 and HsBrf1 indicate conserved regions I, II, and III (Mital et al. 1996). In HsBrf1_v2, the blue region is identical to the corresponding HsBrf1 region. the yeasts Candida albicans, Kluyveromyces lactis, S. well as the C-terminal region present in Brf1. Although pombe, and S. cerevisiae (Khoo et al. 1994). Regions II HsBrf1 is not involved in human U6 transcription, and III are also conserved in the human Brf1 protein (Mi- HsBrf1_v2 has been implicated in U6 transcription be- tal et al. 1996; Andrau et al. 1999). Consistent with the cause when antibodies recognizing all HsBrf1 variants antibody depletion data, the C-terminal domain of were used to deplete extracts, U6 transcription was lost HsBrf2 shows very little, if any, homology with Brf1. and could be specifically restored by addition of material HsBrf2 was isolated through a database search for pro- immunopurified from cells expressing tagged HsBrf1_v2 teins related to TFIIB and to the TFIIB-related segment of (McCulloch et al. 2000). It will be necessary to define the Brf1 (Schramm et al. 2000), as well as through biochemi- composition of this immunopurified fraction to confirm cal purification of a complex, consisting of HsBrf2 and the role of HsBrf1_v2 in U6 transcription. four associated proteins, required for transcription from type 3 promoters (Teichmann et al. 2000). It is clear that Identification of human Bdp1 HsBrf2 itself is specifically required for transcription from type 3, but not types 1 and 2, promoters, but the Figure 4shows the structure of S. cerevisiae Bdp1. It exact role of the HsBrf2-associated factors remains to be contains a domain related to a Myb repeat, identified in determined. Although in one case, U6 transcription in the SWI–SNF and ADA complexes, the transcriptional HsBrf2-depleted extracts could be restored only by addi- corepressor N-Cor, and yeast TFIIIB Bdp1, and therefore tion of the HsBrf2-containing complex immunopurified referred to as the SANT domain (Aasland et al. 1996). from HeLa cells expressing tagged HsBrf2 (Teichmann et The SANT domain is absolutely required for TFIIIC-de- al. 2000), in another case it could be restored by addition pendent (but not TFIIIC-independent, see below) RNA of just HsBrf2 synthesized in E. coli (Schramm et al. polymerase III transcription (Kumar et al. 1997). In addi- 2000). This last observation suggests that the HsBrf2- tion, a region upstream of the SANT domain (indicated associated polypeptides may not be absolutely required in orange in Fig. 4) is required for transcription from for U6 transcription but may contribute to the efficiency linear, but not supercoiled, templates (Kassavetis et al. of the reaction. 1998a). Figure 3 also illustrates the structure of HsBrf1_v2 Human Bdp1 cDNAs were isolated through a combi- (originally named BRF2), a factor encoded by one of at nation of database searches for sequences similar to the least four alternatively spliced BRF1 pre-mRNAs (Mc- yeast Bdp1 SANT domain and library screening Culloch et al. 2000). HsBrf1_v2 lacks the zinc finger do- (Schramm et al. 2000). The structure of the protein en- main and the first repeat that are present in Brf1 and coded by one of these cDNAs (HsBdp1) is shown in Fig- conserved in the other proteins of the TFIIB family, as ure 4. It is highly related to the yeast protein within the GENES & DEVELOPMENT 2599 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez Figure 4. Comparison of the ScBdp1 and HsBdp1 polypeptides. The proteins contain a conserved SANT domain (brown box). The regions upstream and downstream of the SANT domain are also quite conserved, especially a segment upstream of the SANT domain (indicated in orange) that is required for transcription from linear, but not supercoiled, templates. The percentages indicate amino acid identities between ScBdp1 and HsBdp1 in the regions bracketed by dotted lines. HsBdp1, HsBdp1_v2, and HsBdp1_v3 are identical in the colored regions. The repeats extend from amino acids 822 to 1338. HsBdp1 and HsBdp1_v2 diverge after amino acid 1353. HsBdp1 and HsBdp1_v3 diverge after amino acid 684. SANT domain (43% identity) as well as both immedi- The characterization of human TFIIIB has revealed ately upstream, in a region that encompasses the seg- that unlike in S. cerevisiae, where type 3 promoters ap- ment required for transcription from linear DNA tem- parently do not exist and one form of TFIIIB serves all plates (21% identity), and downstream (17% identity). RNA polymerase III promoters (Joazeiro et al. 1994), Outside of these regions, the two proteins are not con- there are at least two forms of TFIIIB in human cells. As served, and the human protein differs from the yeast pro- shown in Figure 5, one of them consists of HsTBP, tein by a striking C-terminal extension containing a HsBrf1, and HsBdp1 and is used by type 2 (and probably number of repeats with potential phosphorylation sites. type 1) promoters. The other consists of HsTBP, HsBrf2, A number of alternatively spliced BDP1 cDNAs have and HsBdp1, and is used by type 3 promoters. Future been isolated (Kelter et al. 2000; Schramm et al. 2000). work may reveal that different spliced variants of Bdp1 Two of these encode strikingly different proteins, which are recruited to different RNA polymerase III promoters are also shown in Figure 4. The longest protein (labeled in vivo. Furthermore, in D. melanogaster cells, the TBP HsBdp1_v2 in the figure) is identical to Bdp1 except that in TFIIIB is replaced by a TBP-related factor called TRF1 the last few amino acids are replaced by a 901-amino- (Takada et al. 2000). Thus, there may be a wide range of acid extension, giving a protein of 2254amino acids. TFIIIB activities in different species containing variants Another cDNA encodes a 725-amino-acid protein of each of the three TFIIIB components. (Bdp1_v3), which contains Bdp1 sequences up to amino In S. cerevisiae, H. sapiens, and D. melanogaster, Brf1 is tightly associated with TBP or TRF1 in solution, as acid 684, followed by a divergent 47-amino-acid exten- sion (Kelter et al. 2000). symbolized by red bars in Figure 5. On the other hand, Which of the alternatively spliced forms of human Bdp1 is weakly associated with the TBP–Brf1 complex in Bdp1 are involved in RNA polymerase III transcription in S. cerevisiae, and very weakly, if at all, in human cells vivo is not clear at present. Depletions of extracts with (Kassavetis et al. 1991; Wang and Roeder 1995; Mital et antibodies directed against regions both upstream and al. 1996; Schramm et al. 2000). Indeed, an association downstream of the SANT domain within the N-terminal between HsBdp1 and HsTBP can only be detected in GST half of human Bdp1 (Schramm et al. 2000), as well as pull-downs (blue bars in Fig. 5; Cabart and Murphy against the repeat region (L. Schramm and N. Hernandez, 2002). Similarly, although HsBrf2 can be shown to asso- unpubl.), debilitate transcription from both type 2 and 3 ciate with HsTBP in GST pull-downs (Cabart and Mur- promoters in vitro, and transcription can be restored by phy 2001, 2002), it is not strongly associated with TBP in addition of recombinant human Bdp1, either full-length HeLa cell extracts (Schramm et al. 2000). Thus, the or truncated downstream of the SANT domain. This sug- TFIIIB components do not always form a stable complex gests that HsBdp1 is generally required for RNA poly- off the DNA. merase III transcription, and that the C-terminal repeats are not required for basal in vitro transcription from na- Functions of TFIIIB ked DNA templates. However, the functional protein present in HeLa cell extracts probably contains the re- In RNA polymerase II transcription, the opening of the peat region, because it can be depleted by antibodies di- transcription bubble that occurs after recruitment of the rected against this region. Perhaps the repeat region per- polymerase is dependent on TFIIE and an ATP-depen- forms a regulatory role not scored in the in vitro tran- dent helicase activity of TFIIH (Holstege et al. 1996; scription assay. Tirode et al. 1999). In contrast, in RNA polymerase III 2600 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Figure 5. Promoter-selective TFIIIB activities. The TFIIIB components required by different classes of promoters in Homo sapiens and Drosophila melanogaster are depicted. Strong (red bars) and weak (blue bars) direct protein–protein associations in solution are indicated. Stippled lines indicate that a direct protein–protein contact has not been demonstrated. DmBdp1 has not been characterized, but a candidate gene has been identified (Schramm et al. 2000). allows it to bind to the mutated TATA box (Strubin and transcription, the opening of the transcription bubble oc- curs in an ATP-independent manner after recruitment of Struhl 1992; Whitehall et al. 1995). The TBP–TATA-box RNA polymerase III by TFIIIB (Kassavetis et al. 1990, complex is then recognized by Brf1 or, in the case of the 1992). The availability, in yeast, of both recombinant human U6 promoter, by Brf2. The similarity of both Brf1 TFIIIB and a transcription system independent of TFIIIC, and Brf2 to TFIIB is very striking, and immediately sug- that is, a system in which a TATA box can recruit TFIIIB gests that the conserved domains of these proteins may directly, has allowed detailed analyses of the functions of perform equivalent functions during assembly of RNA the TFIIIB subunits. These studies have given a detailed polymerase II and III transcription initiation complexes. picture of how TFIIIB recognizes the TATA box and how The reality, however, is more complex. In TFIIB, the core it recruits RNA polymerase III. They have also revealed domain is sufficient for association with the TATA-box– that, remarkably, TFIIIB not only functions to recruit TBP complex. However, recruitment of RNA polymer- RNA polymerase III but also participates in opening of ase II and TFIIF to the TATA-box–TBP–TFIIB complex the transcription bubble. requires the TFIIB zinc-binding domain (Barberis et al. 1993; Ha et al. 1993; Hisatake et al. 1993; Yamashita et al. 1993; Pardee et al. 1998). HsBrf2 resembles TFIIB in that it recognizes the Binding of TFIIIB to the TATA box TATA-box/TBP complex through its TFIIB-related core In promoters consisting of just a TATA box, S. cerevisiae domain (Cabart and Murphy 2001). In contrast, for S. TFIIIB binds to the DNA through recognition of the cerevisiae Brf1, the task of recognizing the TBP–TATA- TATA box by its TBP subunit. Indeed, a mutation in the box complex is performed by two regions of the protein, TATA box that debilitates RNA polymerase III tran- the TFIIB-related N-terminal half as well as the Brf1- scription can be compensated by a mutation in TBP that specific C-terminal half, with the latter playing the ma- alters the DNA-binding specificity of the protein and jor role. Thus, as summarized in Figure 6, a truncated Figure 6. Functional domains of Saccharomyces cerevisiae Brf1. S. cerevisiae Brf1 is depicted, with the locations of the zinc domain, direct repeats in the core, and conserved regions I, II, and III. The brackets below indicate regions of the proteins sufficient for association with the TBP–TATA-box complex (Kassavetis et al. 1998b), TBP alone (Khoo et al. 1994), and the C34 (Khoo et al. 1994; Andrau et al. 1999) and C17 (Ferri et al. 2000) subunits of RNA polymerase III. The black boxes indicate regions where mutations or deletions have a strong negative effect on the associations. The stippled line indicates an association detected only by UV cross- linking. The upstream boundary of the Brf1 region sufficient for interaction with C17 is not precisely defined. GENES & DEVELOPMENT 2601 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ScBrf1 protein retaining just the zinc-binding domain TATA-box–TBP–HsBrf2 complex (Cabart and Murphy and the core associates only very weakly with a TATA- 2002). box–TBP complex. Indeed, the association is so weak that it is only detected by methods such as photochemi- cal cross-linking (Kassavetis et al. 1997, 1998b; Colbert RNA polymerase recruitment by TFIIIB et al. 1998). This weak association appears to involve a Figure 7 shows the known protein–protein contacts be- TBP surface that overlaps or lies near the TFIIB-interact- tween TFIIIB and RNA polymerase III subunits with ar- ing surface in the “stirrup” of the second TBP repeat, rows for contacts identified with human (solid) or yeast because a triple-amino-acid change in TBP that disrupts (hatched) subunits, respectively. Eight RNA polymerase the TFIIB interaction suppresses cross-linking of the N- III subunits can be cross-linked to DNA in a transcrip- terminal half of ScBrf1 to DNA and thus probably asso- tion initiation complex (Bartholomew et al. 1993). Of ciation with the TBP–TATA-box complex (Kassavetis et these, C34, which is part of the three-subunit subcom- al. 1998b). On the other hand, a 110-amino-acid region plex that is required for transcription initiation (Werner encompassing conserved region II within the C-terminal et al. 1993; Wang and Roeder 1997), maps the furthest half of the protein is sufficient for stable association with upstream and can be localized between positions −17 and a TATA-box–TBP complex as well as for recruitment of +6 relative to the transcription start site, in close prox- ScBdp1. Moreover, the hydroxyl radical footprint ob- imity to TFIIIB (Bartholomew et al. 1993). ScBrf1 inter- served with just the C-terminal domain of ScBrf1 is iden- acts in vivo and in vitro with C34, and human Brf1 as- tical to that observed with the full-length protein (Col- sociates with the human homolog of C34, HsRPC39, in bert et al. 1998). Thus, despite the strong conservation of vitro (Werner et al. 1993; Khoo et al. 1994; Wang and the core domains in TFIIB and Brf1, it appears that in Roeder 1997). As shown in Figure 6, ScBrf1 appears to ScBrf1, the function of recognizing the TBP–TATA-box contact C34through three regions: regions II and III complex has been largely transferred to the C-terminal within the Brf1-specific C-terminal domain (Andrau et half of the protein and in particular to conserved region al. 1999), and another region, identified by GST pull- II. This region of ScBrf1 binds the opposite face of the down assays, located within the core region in the TFIIB- TBP–TATA-box complex from TFIIB and recognizes a related N-terminal half of the protein (Khoo et al. 1994). TBP surface that overlaps that recognized by TFIIA (Col- ScBrf1 also contacts the recently identified RNA poly- bert et al. 1998; Kassavetis et al. 1998b; Shen et al. 1998; merase III subunit C17 through the C-terminal half of its for models of the structure of the TBP–DNA–ScBrf1 core region (Ferri et al. 2000). Notably, unlike the zinc- complex, see Colbert et al. 1998; Geiduschek and Kassa- binding domain of TFIIB, the zinc-binding domain of vetis 2001). It will be important to contrast HsBrf1 and ScBrf1 is not required for RNA polymerase recruitment HsBrf2 with ScBrf1 to determine how these TBP-associa- (Kassavetis et al. 1997; Hahn and Roberts 2000). How tion activities have been conserved among the human HsBrf2 contacts RNA polymerase III is not known. It Brf1 and Brf2 proteins. will be highly interesting to determine further which ScBdp1 can associate with a preassembled TBP– parts of the protein are required for assembly with TBP ScBrf1–TATA-box complex but not with a complex lack- and SNAP onto the human U6 promoter and for recruit- ing ScBrf1, and this confers on the yeast TFIIIB–DNA ment of RNA polymerase III. Contacts between Bdp1 complex its striking resistance to salt and heparin. and RNA polymerase III subunits have not been de- ScBdp1 contacts not only ScBrf1 but also TBP, because at scribed, but as shown in Figure 7, human TBP associates least one mutation in yeast TBP prevents association of ScBdp1 without affecting association of ScBrf1 (Colbert et al. 1998). ScBdp1 also contacts DNA because its as- sembly onto the TATA-box–TBP–ScBrf1 complex both requires DNA, and extends the DNA footprint, upstream of the TATA box (Colbert et al. 1998; Shah et al. 1999). Moreover, ScBdp1 can be cross-linked to the DNA at sites upstream of the TATA box (Shah et al. 1999). The binding of ScBdp1 to the TBP–ScBrf1–TATA-box com- plex induces a bend in the DNA between the TATA box and the transcription start site, which is in phase with the bend imposed by TBP on the TATA box (Leveillard et al. 1991; Braun et al. 1992b; Grove et al. 1999). This bending of the DNA has been postulated to contribute to the ScBdp1-dependent stabilization of the TFIIIIB–DNA complex by helping impede sliding of the DNA out of the complex (Grove et al. 1999), a hypothesis consistent Figure 7. Protein–protein contacts between TFIIIB compo- with thermodynamic and kinetic data indicating nents and RNA polymerase III subunits. The solid arrows rep- ScBdp1-dependent kinetic trapping of the DNA (Cloutier resent contacts identified with human subunits, the stippled et al. 2001). In the human system, HsBdp1 has been arrows depict contacts identified with Saccharomyces cerevi- shown to assemble, albeit inefficiently, on a preformed siae subunits. 2602 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors with the HsRPC39 RNA polymerase III subunit in vitro Recruitment factors: TFIIIA (Wang and Roeder 1997). In an in vitro system in which TFIIIB can be recruited directly to a TATA box, TFIIIB on its own is sufficient to recruit RNA polymerase III. In natural RNA polymerase Post-RNA polymerase III recruitment roles for Brf1 III promoters, however, TFIIIB is recruited to the DNA in andBdp1 large part through protein–protein contacts with pro- moter-bound recruitment factors, specifically TFIIIC or In in vitro transcription assays with supercoiled tem- plates, the activities of S. cerevisiae Brf1 and Bdp1 are SNAP . The type 1 5S promoters and type 2 promoters surprisingly resistant to deletions. The N-terminal half both use TFIIIC, but on the 5S promoters TFIIIC is re- of ScBrf1 forms an unstable TFIIIB complex but is nev- cruited through the specificity factor TFIIIA. TFIIIA is ertheless capable of directing TFIIIC-independent tran- the founding member of the C H zinc finger family of 2 2 scription from a TATA box if high amounts of ScBdp1 DNA-binding proteins (Miller et al. 1985) and contains are supplied (Kassavetis et al. 1997). The C-terminal half nine C H zinc fingers. In S. cerevisiae, the only essen- 2 2 on its own shows little or no transcription activity, but tial role of TFIIIA is in the transcription of the 5S RNA when the N-terminal half of ScBrf1 is added in trans, genes, because strains engineered to express the 5S peptides encompassing region II mediate high levels of rRNA from a tRNA-type promoter and lacking TFIIIA transcription. Perhaps most surprising, an ScBrf1 protein are viable (Camier et al. 1995). This may explain in part lacking the first 164amino acids including the zinc- the rapid evolution of TFIIIA: TFIIIA sequences from binding domain and the first TFIIB-related repeat retains various organisms are poorly conserved, even among ver- up to 25% of the activity of full-length ScBrf1 for TFIIIC- tebrates. As an example, human and X. laevis TFIIIAs independent transcription from supercoiled templates in share 61% identity over a 264-amino-acid region—of 423 vitro (Kassavetis et al. 1997). Importantly, however, none and 344 amino acids for the human (Arakawa et al. 1995) of these ScBrf1 truncations function in vivo or for and X. laevis (Ginsberg et al. 1984) proteins, respec- TFIIIC-dependent transcription in vitro. Moreover, they tively—whereas the RNA polymerase II transcription do not function for TFIIIC-independent transcription in factor TFIIB is 94% identical in the two species over its vitro from a linear template, suggesting that they are entire length. TFIIIA binds directly to the ICR of type 1 somehow defective in promoter opening. Indeed, with promoters. TFIIIA also binds to 5S RNA to form the 7S the ScBrf1 protein lacking the first 164amino acids, storage ribonucleoprotein particle (Pelham and Brown RNA polymerase III is recruited on a linear template, but 1980). It is present in massive amounts in immature X. the transcription bubble does not form (Kassavetis et al. laevis oocytes, because they accumulate 5S RNA for 1998a). This is probably caused at least in part by the later use during oogenesis and the first rounds of embry- absence of the ScBrf1 zinc ribbon, because point muta- onic cell division, which occur at a rapid pace in the tions within the zinc domain show defects in promoter absence of transcription. This allowed early on the puri- opening as determined by sensitivity to potassium fication of TFIIIA to near homogeneity; indeed, X. laevis permanganate (Hahn and Roberts 2000). Therefore, in TFIIIA was the first eukaryotic transcription factor to be ScBrf1, the zinc ribbon, which is not required for poly- purified (Engelke et al. 1980) and the first for which a merase recruitment, plays a role at a later stage, during corresponding cDNA was isolated (Ginsberg et al. 1984). promoter opening. Upon binding of X. laevis TFIIIA to the 5S gene, the The ScBdp1 TFIIIB subunit also plays a post-RNA TFIIIA zinc fingers are aligned over the length of the ICR with the C-terminal zinc finger in proximity of the 5 polymerase III recruitment role. Thus, ScBdp1 molecules end, and the N-terminal finger in proximity of the 3 end, lacking the conserved region upstream of the SANT of the ICR (for references, see Paule and White 2000). domain (see Fig. 4) can direct somewhat reduced levels Zinc fingers 1–3, which contact the C box, have been of transcription from supercoiled templates but are reported to contribute most of the binding energy of the inactive with linear templates and fail to generate entire protein (Clemens et al. 1992; Liao et al. 1992). permanganate sensitivity around the transcription start Interestingly, however, like TFIIIA fragments containing site (Kassavetis et al. 1998a). Moreover, ScBdp1 is fingers 1–3, fragments containing fingers 4–9 bind, in dispensable for transcription altogether under conditions in which promoter opening is not required (Kassavetis this case to the A box and intermediate element, with et al. 1999). Upon recruitment of RNA polymerase III, affinities approaching that of the full-length protein the SUP4 tRNA gene promoter opens in two seg- (Liao et al. 1992; Kehres et al. 1997). This observation, as ments, one surrounding the transcription start site and well as the binding behavior of full-length proteins with the other located ∼ 7 bp upstream (Kassavetis et al. 1992). zinc fingers mutated either singly or in pairs, suggest With templates containing preformed bubbles extending that simultaneous binding by all nine TFIIIA zinc fingers from −9 to −5, TBP and ScBrf1 alone are sufficient to DNA requires energetically unfavorable distortions, to recruit RNA polymerase III and direct multiple rounds either in the DNA, the protein, or both. Thus, there is of transcription, although the efficiency is only 5% negative cooperativity between certain zinc fingers such to 10% of that observed with the complete TFIIIB com- that loss of binding by a subset of zinc fingers has only a plex. Thus, ScBdp1 plays an essential role in promoter small negative effect on the overall stability of the com- opening. plex (Kehres et al. 1997). Although TFIIIA on its own is GENES & DEVELOPMENT 2603 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez displaced from DNA upon passage of RNA polymerase 1989). These results suggest a factor consisting of two III, the unusual TFIIIA binding properties may contrib- DNA-binding modules separated by a flexible linker that ute to the resilience of the complete 5S transcription can accommodate variously spaced A and B boxes. complex to repeated passage of the RNA polymerase (Bo- genhagen et al. 1982; Setzer and Brown 1985; Wolffe et al. 1986; Darby et al. 1988; Kehres et al. 1997). Yeast TFIIIC Surprisingly little is known about how TFIIIA recruits S. cerevisiae TFIIIC consists of six subunits, Tfc3/138, TFIIIC to the DNA. X. laevis TFIIIA contains a 14- Tfc4/131/PCF1, Tfc1/95, Tfc6/91, Tfc8/60, and Tfc7/ amino-acid domain located C-terminal of the ninth zinc 55, all of which have been cloned and shown to be es- finger, and thus located toward the 5 end of the ICR in sential for cell viability (Willis et al. 1989; Swanson et al. the TFIIIA/5S gene complex, that is dispensable for DNA 1991; Lefebvre et al. 1992; Marck et al. 1993; Arrebola et binding but essential for transcription (Mao and Darby al. 1998; Manaud et al. 1998; Deprez et al. 1999). Five S. 1993). In S. cerevisiae TFIIIA, a hydrophobic segment pombe proteins have recently been identified by BLAST within an 84-amino-acid region located between zinc searches with S. cerevisiae TFIIIC subunits as the query, fingers 8 and 9 is similarly required for cell viability and and four of them confirmed as TFIIIC subunits by im- transcription but not for DNA binding (Rowland and Se- munoaffinity purification of a functional TFIIIC com- gall 1998). These protein domains may play a role in the plex from cells expressing the tagged polypeptides recruitment of TFIIIC. (Huang et al. 2000). These four subunits, referred to as Sfc1, Sfc3, Sfc4, and Sfc6, are orthologs of S. cerevisiae Tfc1, Tfc3, Tfc4, and Tfc6, respectively, as shown in Recruitment factors: TFIIIC Table 2. The fifth one, referred to as Sfc9, shares se- The transcription factor TFIIIC is capable of recognizing quence homology with the S. cerevisiae Tfc8 subunit the TFIIIA–ICR complex on type 1 5S RNA promoters within a short C-terminal segment and may, therefore, and the variously spaced A and B boxes on type 2 tRNA correspond to the S. pombe ortholog of Tfc8. The iden- promoters. The structure of the factor is uniquely tification of these S. pombe subunits is very interesting adapted to perform these tasks. Proteolysis studies indi- because, as detailed below, it clarifies in some cases the cate that S. cerevisiae TFIIIC consists of two domains relationship between S. cerevisiae and human TFIIIC separated by a flexible linker, one of which, designated subunits (Huang et al. 2000). , binds strongly to the B box and the other, designated , binds weakly to the A box (Marzouki et al. 1986). Depending on the distance separating the A and B boxes, Human TFIIIC the factor is visualized by scanning electron microscopy as either two tightly packed or two clearly separated The human TFIIIC fraction contains several activities, globular domains of roughly similar sizes (Schultz et al. some of which are not yet completely defined. These are Table 2. Saccharomyces cerevisiae TFIIIC components andorthologues in Schizosaccharomyces pombe and Homo sapiens S. cerevisiae S. pombe TFIIIC TFIIIC H. sapiens TFIIIC2a Comments Tfc3/ Sfc3 TFIIIC220/TFIIIC Tfc3 and Sfc3 are related, but neither shows sequence similarity to TFIIIC220. Tfc3 cooperates with Tfc6 for binding to DNA. Fragments of TFIIIC220 and TFIIIC110 form a subcomplex capable of binding to the B-box. Tfc4/ /PCF1 Sfc4TFIIIC102/TFIIIC Tfc4protrudes upstream of the start site. TPRs. Tfc4contacts ScBrf1, ScB dp1, and ABC10. Most conserved of the TFIIIC subunits. TFIIIC102 associates with HsBrf1, HsTBP, TFIIIC63. Tfc1/ Sfc1 TFIIIC63/TFIIIC tRNA A-box binding. Tfc1 and Tfc7 associate and can form a distinct complex. TFIIIC63 associates with TFIIIC102, HsBrf1, HsTBP, and HsRPC62. Tfc6 ( ) Sfc6 TFIIIC110/TFIIIC Binds terminator. HMG-I and HMG-Y motifs, WD-40 repeats. Similarity between Tfc6 and TFIIIC110 apparent only through Sfc6. Tfc6 cooperates with Tfc3 for binding to DNA. TFIIIC110 and TFIIIC220 form a subcomplex capable of binding to the B-box. Full-length TFIIIC110 absent in TFIIIC2b. TFIIIC110 possesses HAT activity. Tfc8 ( ) Sfc9 TFIIIC90/TFIIIC Tfc8 bridges  and  domains as well as TFIIIB. Associates with ScTBP. 60 B A Similarity of Tfc8 and Sf9 limited to short C-terminal segment. No similarity between TFIIIC90 and the yeast proteins, but TFIIIC90 binds to TFIIIC220, 110, 63, HsBrf1, HsRPC62, and HsRPC39, and thus may be a functional homolog of the yeast proteins. TFIIIC90 displays HAT activity for histone H3 Lys 14. Tfc7 ( ) none none tRNA A box binding. Tfc7 and Tfc1 associate and can form distinct complex. 2604 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors summarized in Figure 8. The TFIIIC fraction was origi- TFIIIC110/TFIIIC, and TFIIIC90/TFIIIC (see Table nally separated into two fractions, TFIIIC1 and TFIIIC2, 2), whereas TFIIIC2b, which represents 10%–20% of to- that were both required for transcription from the Ad2 tal TFIIIC2 in actively dividing HeLa cells, lacks the VAI gene (Yoshinaga et al. 1987). The TFIIIC1 fraction is TFIIIC110 subunit and appears to contain a 77-kD sub- only partially defined and is discussed further below. unit absent in TFIIIC2a (see Fig. 8; Yoshinaga et al. 1989; The activity in the TFIIIC2 fraction seems to correspond Kovelman and Roeder 1992; Sinn et al. 1995). The nature in part to that of the yeast TFIIIC complex and resides in of the 77-kD subunit is not clear; it is not recognized by a complex of five polypeptides referred to as TFIIIC2 or antibodies generated against the last 595 amino acids of TFIIIC2a (Yoshinaga et al. 1989; Kovelman and Roeder TFIIIC110, suggesting that it either corresponds to an 1992). Human TFIIIC2a has, however, some added ac- unrelated protein or to a TFIIIC110 fragment devoid of tivities compared with S. cerevisiae TFIIIC (see Table 2). epitopes recognized by the antibody. Thus, in cell lines expressing a tagged TFIIIC2a subunit, The TFIIIC220 subunit functionally corresponds to TFIIIC2a can be purified by immunoprecipitation as part the S. cerevisiae Tfc3 subunit because, like Tfc3, it rec- of a larger complex called holo-TFIIIC (see Fig. 8), which ognizes the B box (see below; Table 2). Very strikingly, is described together with the TFIIIC1 fraction further however, it shares no sequence similarity with either S. below (Wang and Roeder 1998; Wang et al. 2000). Holo- cerevisiae Tfc3 or S. pombe Sfc3p (L’Etoile et al. 1994; TFIIIC can bind to chromatin templates, relieves the Lagna et al. 1994; Huang et al. 2000). Similarly, the hu- chromatin-mediated repression of RNA polymerase III man TFIIIC90 protein (Hsieh et al. 1999a) has no obvious transcription from a tRNA gene, and displays histone sequence homolog in S. cerevisiae, but the set of TFIIIC acetyltransferase (HAT) activity (Kundu et al. 1999), and TFIIIB subunits it interacts with suggest that it is the which can be attributed to the intrinsic HAT activity of functional equivalent of Tfc8 (see below). TFIIIC90 has, at least two of the TFIIIC2a subunits (Hsieh et al. 1999a; however, the added property of an intrinsic HAT activity Kundu et al. 1999). for both free and nucleosomal histone H3, and preferen- Besides the active TFIIIC2a complex, the TFIIIC2 frac- tially acetylates histone H3 Lys 14(Hsieh et al. 1999a). tion contains a transcriptionally inactive TFIIIC2 com- The TFIIIC110 subunit corresponds to the yeast Tfc6p plex designated TFIIIC2b (Fig. 8; Kovelman and Roeder protein, although the similarity between the two pro- 1992). TFIIIC2a consists of five subunits referred to as teins is apparent only when compared with the S. pombe TFIIIC220/TFIIIC, TFIIIC102/TFIIIC, TFIIIC63/TFIIIC, ortholog (Huang et al. 2000). Like TFIIIC90, TFIIIC110 Figure 8. Schematic representing the components identified in the human TFIIIC fraction. Holo-TFIIIC was purified by immunoaf- finity from a cell line expressing tagged TFIIIC220 (Wang and Roeder 1998). It is likely, therefore, that it contains TFIIIC2b as well as TFIIIC2a, although this has not been directly demonstrated. See text for references. GENES & DEVELOPMENT 2605 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez has intrinsic HAT activity, and acetylates free and cross-linking (Gabrielsen et al. 1989; Yoshinaga et al. nucleosomal histones H3 and H4as well as nucleosomal 1989; Bartholomew et al. 1990, 1991; Braun et al. 1992a; histone H2B (Kundu et al. 1999). In contrast to the hu- Kovelman and Roeder 1992) and protein–protein associa- man TFIIIC subunits just described, the TFIIIC102 and tion experiments (Shen et al. 1996; Manaud et al. 1998; TFIIIC63 subunits are very clearly related to their yeast Hsieh et al. 1999a,b), TFIIIC models such as those shown counterparts, Tfc4and Tfc1, respectively (Hsieh et al. in Figure 9 can be drawn. In Figure 9, the main sites of 1999b). Thus, human TFIIIC2a is surprisingly divergent cross-linking between yeast subunits and DNA are indi- from S. cerevisiae TFIIIC in all but two of its subunits. cated with dots. Known protein–protein contacts among As detailed further below, these two conserved subunits TFIIIC subunits are indicated by dashes according to are located close to the transcription start site and inter- whether they were demonstrated with human (black) or act directly with TFIIIB subunits. yeast (gray) subunits. On the SUP4 tRNA gene, the S. cerevisiae Tfc3 sub- unit cross-links primarily just upstream of the B box and Assembly of a stable initiation complex on type 1 Tfc6 cross-links at the end of the gene (Bartholomew and 2 promoters et al. 1990, 1991). The two proteins probably cooper- ate to bind to DNA because a mutation in Tfc6 can al- TFIIIC performs at least three functions: It recognizes leviate the binding defect of a Tfc3 mutant (Arrebola et promoter elements, either directly in the case of type 2 al. 1998). Consistent with Tfc3 and Tfc6 corresponding promoters or with the help of TFIIIA in the case of type to human TFIIIC220 and TFIIIC110, respectively, hu- 1 promoters; it recruits TFIIIB; and it contributes to the man TFIIIC220 can be cross-linked to the B box (Yoshi- recruitment of RNA polymerase III. For all of these func- naga et al. 1989; Kovelman and Roeder 1992). Moreover, tions, we have a good idea of which of the TFIIIC sub- TFIIIC220 does not bind DNA on its own, but it is part units are involved. of TFIIIC subassemblies generated by proteinase C cleav- age during poliovirus infection that are still capable of Binding of TFIIIC to type 2 and type 1 promoters binding to DNA (Clark et al. 1991). Some of these sub- A recombinant complex has not yet been reconstituted complexes consist of just the N-terminal 83 kD of either from yeast or human cells, but from both photo- TFIIIC220 associated with the TFIIIC110 subunit or a Figure 9. The position of the various Sc TFIIIC and TFIIIB subunits on an SUP4 tRNA and 5S gene as determined by cross-linking and protein–protein association experiments (see text) are indicated. For the TFIIIC subunits, the names of both the Saccharomyces cerevisiae and the human subunits are indicated. The positions of the start site, end of the gene, and A, B, and C boxes are indicated. A bend in the DNA at the TFIIIB binding site is not illustrated in the figure. The colored dots indicate major cross-linking sites of the subunit of matching color to the DNA. The Tfc8 subunit is shown as an elongated shape extending over most of the TFIIIC complex because it appears to bridge the TFIIIC  and  domains as well as TFIIIB. The black and gray rectangles illustrate protein–protein A B contacts identified with human and S. cerevisiae TFIIIC subunits, respectively. Protein–protein contacts between TFIIIC and TFIIIB subunits are summarized in Figure 10A. 2606 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors fragment thereof, indicating that the DNA-binding do- to sites around and upstream of the start site of tran- main of TFIIIC220 lies within the N-terminal region of scription (Braun et al. 1992a). the protein and that TFIIIC220 and TFIIIC110 are suffi- cient to generate the DNA-binding surface of the TFIIIC2a complex (Shen et al. 1996). Note that an essen- TFIIIC recruitment of TFIIIB tial role for the TFIIIC110 subunit in DNA binding im- plies that the TFIIIC2b complex, which can bind DNA The role of TFIIIC in the recruitment of TFIIIB has been specifically, contains either an unrecognized TFIIIC110 intensively studied in S. cerevisiae. Experiments in fragment or another protein (e.g., the 77-kD protein) pro- which various TFIIIB components were added sequen- viding the TFIIIC110 DNA-binding function. tially to a TFIIIC–DNA complex suggest that the TFIIIC– The yeast Tfc1 and Tfc7 subunits have strong cross- DNA complex interacts initially with the Brf1 compo- links within and near the 3 end of the A box, respec- nent of TFIIIB (Kassavetis et al. 1992), most probably tively (Bartholomew et al. 1990, 1991). Tfc7 interacts through the Tfc4subunit, which protrudes upstream of directly with Tfc1 through its C-terminal half, and the the transcription start site (see Fig. 9). Like the interac- two proteins are not only part of TFIIIC but also form a tion of TFIIIC with its bipartite binding site on DNA, the separate complex in yeast cells (Manaud et al. 1998). interaction of TFIIIC with TFIIIB is highly flexible. Thus, Tyr Tfc8 does not cross-link to DNA. When TFIIIC is treated when TFIIIB is recruited by TFIIIC on SUP4 tRNA with protease, Tfc8 is found in the  domain as deter- gene constructs with TATA boxes inserted at various mined by antibody supershift experiments. In addition, positions upstream of the A box and TFIIIC is subse- however, the temperature sensitivity observed with quently stripped from the DNA by heparin treatment, strains expressing an epitope tagged version of Tfc8 is the upstream and downstream borders of the footprint specifically suppressed by overexpression of Tfc1, which occasioned by the remaining TFIIIB vary according to the resides in the  domain. It is also suppressed by over- position of the TATA box. On the same constructs, the expression of TBP and ScBdp1, and Tfc8 associates with footprint of TFIIIC alone is invariant, but the footprint TBP in vitro (Deprez et al. 1999). Human TFIIIC90 is attributable to TFIIIC (heparin-sensitive) in the presence thought to be the functional homolog of Tfc8 because it of TFIIIB extends to cover the interval between the bor- interacts with TFIIIC220, TFIIIC110, and TFIIIC63, as ders of the variant TFIIIB footprint and that of the invari- well as with the TFIIIB subunit HsBrf1 (Hsieh et al. ant TFIIIC footprint observed with TFIIIC alone. Thus, 1999a). Thus Tfc8/TFIIIC90 appears to bridge the TFIIIC the placement of TFIIIB is codirected by the TATA box and  domains as well as TFIIIB, and is represented, (and thus probably the TBP subunit of TFIIIB) and B A therefore, as extending over the entire gene in Figure 9. TFIIIC. Furthermore, because the interval protected is The Tfc4subunit cross-links to sites around and up- the region to which the Tfc4subunit of TFIIIC cross- stream of the transcription start site (Bartholomew et al. links, these results suggest that Tfc4can extend to ac- 1990, 1991) and directly contacts both the ScBrf1 and commodate various spacings between TFIIIB and TFIIIC ScBdp1 subunits of TFIIIB (Khoo et al. 1994; Chaussivert (Joazeiro et al. 1996). et al. 1995; Rüth et al. 1996; Dumay-Odelot et al. 2002). Tfc4contains 11 copies of the tetratricopeptide repeat Its location on the DNA is consistent with the human (TPR) in four blocks of 5, 4, 1, and 1 repeats (Marck et al. ortholog, TFIIIC102, interacting with TFIIIC63, TBP, 1993; Rameau et al. 1994; Dumay-Odelot et al. 2002). and HsBrf1 (Hsieh et al. 1999b). Tfc1/TFIIIC63 is shown TPRs are found in a large number of proteins including extending upstream of the transcription start site be- subunits of the anaphase-promoting complex and the cause the human subunit, like TFIIIC102, contacts transcription repressor Ssn6. The crystal structures of HsBrf1 and TBP (Hsieh et al. 1999b). several TPR domains indicate that each TPR consists of On the 5S RNA genes, S. cerevisiae TFIIIA cross-links a pair of antiparallel -helices, and that adjacent TPRs strongly to the A box and much more weakly to posi- are packed in regular series of antiparallel -helices (Das tions as far upstream as +20 and as far downstream as et al. 1998; Scheufler et al. 2000). Truncated Tfc4pro- +127, and thus extends over a large portion of the gene teins encompassing the N-terminal, middle, and C-ter- (only the cross-links on the A box are shown in Fig. 9; minal third of the protein including the first (repeats Braun et al. 1992a). TF3C appears shifted downstream as 1–5), second (repeats 6–9), and last (repeats 10 and 11) compared with its position in the tRNA gene, with a blocks of TPRs, respectively, all bind to ScBrf1 in a GST main cross-link at the 3 end of the C box and another pull-down assay (Khoo et al. 1994). Quantitative in vitro one further downstream. As in the tRNA gene, the Tfc6 equilibrium binding assays with various truncated forms subunit cross-links at the end of the gene. There is no of Tfc4also detect several fragments capable of binding indication that the Tfc7 subunit contacts DNA in the 5S to ScBrf1, of which the two with the highest affinities gene, but the Tfc1 subunit cross-links strongly upstream extend from the N terminus of the protein to the end of of the A box. Thus, the 5S A box is clearly not the func- repeat 5, and from repeat 6 to repeat 9 (Moir et al. 2002a). tional equivalent of the tRNA A box. Rather, the func- Interestingly, both fragments have higher affinities for tional equivalent of the tRNA A box in the 5S gene is the ScBrf1 than a larger fragment extending from the N ter- site of Tfc1 cross-linking, which in both genes occurs minus to repeat 9 (Moir et al. 2002a), consistent with the about 30 nt downstream of the transcription start site. observation that in a yeast two-hybrid assay, a similar As in the SUP4 tRNA gene, the Tfc4subunit cross-links long Tfc4fragment shows weaker association than frag- GENES & DEVELOPMENT 2607 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ments shorter at the C terminus (Chaussivert et al. (Hsieh et al. 1999b). Tfc8 associates with ScBdp1 and 1995). These results suggest that there is autoinhibition ScTBP (Deprez et al. 1999), and the human ortholog for ScBrf1 binding within Tfc4, which is probably re- TFIIIC90 with HsBrf1 (Hsieh et al. 1999a). TFIIIC63 as- lieved by conformational changes during binding. sociates with both HsBrf1 and HsTBP (Hsieh et al. Conformational changes within Tfc4are also sug- 1999b). Thus, there is a network of protein–protein con- gested by an analysis of two dominant Tfc4mutations, tacts between TFIIIC and TFIIIB, all of which may par- PCF-1 and PCF-2, that map within repeats 1–3. These ticipate in the recruitment of TFIIIB during transcription mutations increase transcription both in vivo and in initiation. vitro by increasing the recruitment of TFIIIB, specifically the binding of Tfc4to ScBrf1 (Rameau et al. 1994; Moir TFIIIC contacts RNA polymerase III et al. 1997, 2002b). For PCF1-1, biochemical studies in- dicate that this effect is achieved via a conformational Several of the TFIIIC subunits have been shown to in- change in Tfc4that overcomes autoinhibition in the teract directly with RNA polymerase III subunits, and ScBrf1 binding reaction (Moir et al. 2000, 2002a,b). Thus, these interactions are depicted in Figure 10B with solid the TPRs appear to be involved in conformational arrows for associations demonstrated with human sub- changes that promote association with ScBrf1 and ac- units and hatched arrows for associations demonstrated commodate the variable placement of TFIIIB. The posi- with S. cerevisiae subunits. The Tfc4TFIIIC subunit in- tive effect of the mutations indicates that the recruit- teracts with the RNA polymerase III C53 subunit in a ment of TFIIIB is a limiting step in vivo, and that the two-hybrid assay (Flores et al. 1999), and with the con- conformational change may therefore serve a regulatory served C-terminal domain of the universal RNA poly- role. merase subunit ABC10 in both a yeast two-hybrid assay Other associations have been observed between iso- and in vitro. This latter interaction is likely to be func- lated TFIIIC and TFIIIB subunits. These are symbolized tionally significant because a thermosensitive mutation in Figure 10A with arrows, according to whether the as- within the C-terminal domain of ABC10 that weakens sociation was demonstrated with human (solid) or yeast the interaction can be rescued in an allele-specific man- (hatched) TFIIIC subunits. As just described, Tfc4asso- ner by overexpression of a variant form of Tfc4that ciates with ScBrf1, and it also associates with ScBdp1 strengthens the interaction (Dumay et al. 1999). Among (Khoo et al. 1994; Chaussivert et al. 1995; Rüth et al. the human TFIIIC subunits, the TFIIIC90 protein inter- 1996; Dumay-Odelot et al. 2002). The human ortholog acts with HsRPC62 and HsRPC39, which are both part TFIIIC102 associates with both HsBrf1 and HsTBP of the subcomplex of RNA polymerase III subunits re- Figure 10. Protein–protein contacts between TFIIIC and TFIIIB subunits, and TFIIIC and RNA polymer- ase III subunits. Contacts as determined either by in vitro or yeast two-hybrid assays are depicted. Ge- netic interactions have not been included. Solid ar- rows depict interactions shown with human pro- teins, stippled arrows depict interactions shown with Saccharomyces cerevisiae proteins. See text for references. (A) Protein–protein contacts between TFIIIC and TFIIIB subunits. (B) Protein–protein con- tacts between TFIIIC and RNA polymerase III sub- units. 2608 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors quired specifically for transcription initiation, and and SNAP45 can each associate independently with TFIIIC63 associates with HsRPC62 in vitro (Hsieh et al. SNAP190, and SNAP43 can associate independently with 1999a). These TFIIIC–RNA polymerase III interactions SNAP50. SNAP43 can also associate with SNAP190, but emphasize that TFIIIC not only recruits TFIIIB but may in coimmunoprecipitations of in vitro translated pro- also participate in the recruitment of RNA polymerase teins, this association is not detectable unless SNAP19 is III. Thus, although TFIIIB is sufficient to recruit RNA present. This suggests that SNAP43 has weak contacts polymerase III, in the cell both TFIIIB and TFIIIC are with both SNAP190 and SNAP19, only the sum of which likely to work in concert to recruit the RNA polymerase. is measurable by the stringent coimmunoprecipitation assay (Henry et al. 1998b). However, partial complexes containing SNAP190 or a truncated SNAP190, SNAP43, Recruitment factors: SNAP and SNAP50 can be assembled both in insect cells in- fected with recombinant baculoviruses (Mittal et al. Type 3 promoters do not contain binding sites for TFIIIA 1999) and from subunits overexpressed in E. coli (Ma and and/or TFIIIC2, and, indeed, they do not require TFIIIC2 Hernandez 2000, 2002), indicating that in these situa- for assembly of a transcription initiation complex. Thus, tions SNAP19 is dispensable for complex assembly. depletion of a transcription extract with antibodies di- The domains involved in protein–protein contacts are rected against the DNA-binding subunit of TFIIIC2, summarized in Figure 11B. Within SNAP190, amino ac- TFIIIC220, affects transcription from the 5S, tRNA, and ids 84–133 are sufficient for association with SNAP19 VAI genes but not from the 7SK and U6 alone, and with SNAP43 together with SNAP19. This snRNA genes (Lagna et al. 1994). Instead, the gene-ex- SNAP190 region, and the N-terminal part of SNAP19, ternal PSE of type 3 promoters recruits the multisubunit are likely to form -helices and may be involved in a complex SNAP . SNAP is a complex containing five c c coiled-coil type of interaction with each other. In different subunits, SNAP190 (Wong et al. 1998), SNAP43, amino acids 164–268 are sufficient for associa- SNAP50/PTF (Bai et al. 1996; Henry et al. 1996), tion with SNAP190 together with SNAP19, and amino SNAP45/PTF (Sadowski et al. 1996; Yoon and Roeder acids 1–163 are sufficient for association with SNAP50. 1996), SNAP43/PTF (Henry et al. 1995; Yoon and Thus, these two association domains in SNAP43 are Roeder 1996), and SNAP19 (Henry et al. 1998b). In the completely separable. The SNAP190 region required for human system, SNAP is involved in transcription of interaction with SNAP45 lies between amino acids 1281 snRNA genes by both RNA polymerases II and III, as depletion of endogenous SNAP from transcription ex- and 1393. The SNAP190 regions required for association tracts debilitates U1 and U6 transcription and transcrip- with SNAP19/SNAP43 and with SNAP45 thus lie at op- tion can be restored in both cases by addition of highly posite ends of the linear molecule (Ma and Hernandez purified recombinant SNAP (Henry et al. 1998b). 2000). The subunit–subunit contacts within SNAP have SNAP binds specifically to the PSE. This binding is c c been determined by reconstitution of partial complexes mediated in part by an unusual Myb domain extending and coimmunoprecipitations of various subsets of in from amino acids 263 to 503 within SNAP190, with four vitro translated full-length or truncated SNAP subunits and one-half Myb repeats designated the Rh (R half), Ra, (Henry et al. 1996, 1998a,b; Wong et al. 1998; Mittal et Rb, Rc, and Rd repeats (see Fig. 11B; Wong et al. 1998). al. 1999; Ma and Hernandez 2000, 2002). Figure 11A Indeed, a SNAP190 segment consisting of just the Myb shows the general architecture of the complex. SNAP19 domain can bind to the PSE (Wong et al. 1998), and in the Figure 11. Subunit–subunit interactions within SNAP .(A) For simplicity, the complex is shown as containing one copy of each subunit, but the stoichiometry of the SNAP subunits has not been determined. (B) The domains of the various subunits sufficient for association with other subunits in a coimmunoprecipitation assay. The TBP recruitment region 1 (TRR1), the Myb domain with the half repeat (Rh) followed by four repeats (Ra, Rb, Rc, and Rd), and the Oct-1 interacting region (OIR) are shown. GENES & DEVELOPMENT 2609 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez context of the full complex, the last two Myb repeats (Rc and Rd), but not the first two and one-half (Rh, Ra, and Rb), are required for binding to the PSE (Mittal et al. 1999). However, even though the Myb domain is re- quired, it is not sufficient. Thus, although a SNAP190 segment consisting of just the Myb domain binds DNA, larger SNAP190 segments do not. The smallest subas- sembly of SNAP subunits characterized that binds spe- cifically to DNA consists of SNAP190 amino acids 84– 505, SNAP43 amino acids 1–268, and SNAP50 (complex #8; Ma and Hernandez 2000, 2002). Consistent with the requirement for parts of SNAP190 and SNAP50 for DNA binding, UV cross-linking experiments suggest that both SNAP190 (Yoon et al. 1995) and SNAP50 (Henry et al. 1996) are in close contact with DNA. Figure 12. Human U6 transcription initiation complex. TBP (yellow) and SNAP (orange) bind cooperatively to the DNA, presumably through a direct protein–protein contact that in- Assembly of a stable transcription complex on the type volves a 50-amino-acid segment within the N-terminal region 3 U6 snRNA promoter of SNAP190. TBP also binds cooperatively with Brf2. SNAP and the Oct-1 POU domain (blue) bind cooperatively to DNA, As noted above, the type 3 promoters such as the U6 through a direct protein–protein contact involving a glutamic promoter contain, in addition to core sequences, a DSE acid at position 7 within the POU domain (blue triangle) and a that serves to enhance transcription from the core pro- lysine at position 900 within SNAP190 (red triangle). This di- moter (see Fig. 1). The DSE can contain a number of rect protein–protein interaction is mediated by a positioned protein-binding sites, but two of them are almost invari- nucleosome (green) that brings into close proximity the octamer sequence and the PSE. Adapted from Zhao et al. (2001). See text ably an SPH (also called NONOCT) element, which re- for references. cruits the transcription factor STAF (also called SPH- binding factor or SBF), and an octamer sequence, which recruits the transcription factor Oct-1 (for reviews, see Hernandez 1992, 2001). is efficiently recruited to the DNA through at least two STAF is a zinc finger protein, and Oct-1 is a founding cooperative binding interactions. SNAP binds coopera- member of the POU-domain protein family. Both pro- tively with TBP, which like SNAP binds poorly to DNA teins contain two types of activation domains, one type on its own, and with the Oct-1 POU domain (Murphy et that specifically activates mRNA-type RNA polymerase al. 1992; Mittal et al. 1996; Mittal and Hernandez 1997). II promoters and another that specifically activates both In cooperative binding of SNAP and TBP, both factors type 3 RNA polymerase III promoters and the very simi- recruit each other to the DNA. Mini-SNAP is also able lar RNA polymerase II snRNA promoters (Tanaka et al. to bind cooperatively with TBP, although in this case, 1992; Das et al. 1995; Schuster et al. 1998). The presence cooperative binding results mainly in the recruitment of of the latter type of activation domains in STAF and TBP, because mini-SNAP binds efficiently to DNA on Oct-1 supports the idea that these proteins activate type its own (Mittal et al. 1999). Within the context of mini- 3 promoters (as well as RNA polymerase II snRNA pro- SNAP , a 50-amino-acid segment within the N-terminal moters) in the cell. Indeed, both proteins have been lo- region of SNAP190, called TRR1 (TBP recruitment re- calized to snRNA promoter sequences in vivo by chro- gion 1; see Fig. 11) is required for cooperative binding matin immunoprecipitation experiments (Zhao et al. with TBP. Interestingly, mini-SNAP complexes lacking 2001; Mach et al. 2002). Thus, as for the core promoter these 50 amino acids are active for transcription, sug- sequences of type 3 promoters, the key factors recruited gesting that during assembly of the U6 transcription ini- by the DSE are well characterized and are available in tiation complex, there are redundant mechanisms to re- recombinant form. cruit TBP to the DNA. Indeed, TBP is efficiently re- The identification of the factors binding directly to cruited to the DNA by cooperative binding with HsBrf2 type 3 promoter elements has allowed the characteriza- to form a complex containing mini-SNAP , TBP, and tion of their assembly onto a human U6 promoter. Our HsBrf2 (Ma and Hernandez 2002), and TBP is therefore present understanding of this process is summarized in shown contacting both SNAP and HsBrf2 in Figure 12. Figure 12. SNAP binds poorly to DNA on its own. In- It remains possible that in the absence of the DSE, co- terestingly, however, a partial complex lacking the C- operative binding of TBP and SNAP is essential to re- terminal two-thirds of the largest subunit of SNAP as cruit the full SNAP to the DNA, which, unlike mini- well as SNAP45, referred to as mini-SNAP , binds more SNAP , binds poorly to the PSE. In the presence of the efficiently to the DNA (Mittal et al. 1999). This implies DSE, however, the full SNAP can be recruited to the that the C-terminal two-thirds of SNAP190 and/or DNA by the Oct-1 POU domain. SNAP45, which associates with the C-terminal portion The Oct-1 POU domain is a bipartite DNA-binding of SNAP190, somehow down-regulate binding of the domain consisting of two helix–turn–helix-containing complex to DNA. On the U6 promoter, however, SNAP DNA-binding modules: an N-terminal POU-specific 2610 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors (POU ) domain and a C-terminal POU-homeo domain The recruitment of SNAP by the Oct-1 POU domain S c (POU ), joined by a flexible linker (Herr and Cleary contributes to transcription activation in vitro. There- 1995). The POU domains of Oct-1 and the pituitary tran- fore, Oct-1 is likely to activate snRNA gene transcrip- scription factor Pit-1 are ∼ 50% identical, but only the tion in vivo not only through its transcription activation Oct-1 POU domain is capable of recruiting SNAP to the domains but also through its DNA-binding domain. This PSE. This difference in activity is due to only one of the activation can be compared to activation in prokaryotes many amino acid differences between the two proteins, a by the  cI repressor, which activates transcription from the  P glutamic acid at position 7 in the Oct-1 POU domain promoter by favoring open complex formation S RM changed to an arginine at the corresponding position in through direct protein–protein contacts between its the Pit-1 POU domain. Thus, an E7R mutation in Oct-1 DNA-binding domain and RNA polymerase (for refer- does not affect Oct-1 binding to the octamer sequence ences, see Ford et al. 1998). Strikingly, the DNA-binding but inhibits cooperative binding with SNAP , and an domain of the  cI repressor is structurally similar to the R7E mutation in Pit-1 imparts to this protein the ability Oct-1 POU domain, and the RNA polymerase subunit to bind cooperatively with SNAP (Mittal et al. 1996). contacted by the cI DNA-binding domain is the sub- Oct-1 associates with SNAP190, the largest subunit of unit, which is, like SNAP , a core promoter binding fac- SNAP , both in a yeast one-hybrid assay and in an elec- tor. This illustrates how E. coli and human cells can use trophoretic mobility shift assay (Ford et al. 1998; Wong similar protein–protein contacts to activate transcrip- et al. 1998). This suggested that cooperative binding of tion. the Oct-1 POU domain and SNAP may result from a The Oct-1–SNAP interaction was originally charac- c c protein–protein contact between the POU domain and terized with U6 promoter probes in which the octamer SNAP190. Indeed, a small domain of SNAP190 extend- sequence had been placed close to the PSE. The exact ing from amino acids 869 to 912 and labeled OIR (Oct- distance separating the octamer and the PSE was not 1-interacting region) in Figure 11 above is sufficient for important, but not surprisingly, cooperative binding was association with the Oct-1 POU domain in an electro- not observed on the natural promoter, where the two phoretic mobility shift assay. Strikingly, the OIR of sites are separated by ∼ 150 bp. This suggested that in the SNAP190 displays sequence similarity with the Oct-1 natural U6 promoter the two sites may somehow be POU-domain-interacting region present in OBF-1/ brought into close proximity for cooperative binding. In- OCA-B (Ford et al. 1998). OBF-1 is a B-cell-specific coac- deed, as shown in Figure 12, the human U6 promoter tivator of Oct-1, which recognizes Oct-1 bound to an harbors a positioned nucleosome between the octamer octamer sequence and provides a strong activation do- sequence and the PSE, both in vivo as determined by main of mRNA gene transcription. As with SNAP190, micrococcal nuclease sensitivity of the chromatin and in the association of OBF-1 with the Oct-1 POU domain is vitro upon assembly of a U6 template into nucleosomes sensitive to the E7R mutation in Oct-1 (Babb et al. 1997), (Stunkel et al. 1997; Zhao et al. 2001). The positioned and, indeed, the Oct-1 POU E7 is involved in a hydrogen nucleosome promotes activation by the Oct-1 POU do- bond with a lysine in OCA-B as shown in the crystal main from its natural binding site as well as cooperative structure of an OCA-B peptide associated with an Oct-1 binding of Oct-1 and SNAP . This cooperative binding is POU/octamer sequence complex (Chasman et al. 1999). affected by the Oct-1 E7R and the SNAP190 K900E mu- Thus, two proteins with different tissue distribution, tations, but is restored to significant levels when the two function, and primary structure nevertheless share a mutant factors are combined (Zhao et al. 2001). Thus, common Oct-1 binding domain, and probably contact the positioned nucleosome mediates a direct protein– overlapping surfaces in the Oct-1 POU domain (Ford et protein contact between the Oct-1 POU domain and al. 1998). SNAP , and this contact is the same as the one charac- Within the SNAP190 OIR, mutation of Lys 900 to a terized on probes containing closely spaced octamer and glutamic acid (K900E mutation) inhibits cooperative PSE. This provides a striking example of a nucleosome, binding of SNAP with the Oct-1 POU domain. Very the structural unit of chromatin, activating transcription significantly, however, if SNAP carrying the K900E mu- by serving an architectural role and thus allowing coop- tation within SNAP190 is combined with the Oct-1 erative interactions of factors binding to distant sites. POU domain carrying the E7R mutation, significant co- The assembly of the U6 transcription complex illus- operative binding is restored (Ford et al. 1998). The iso- trates how a network of cooperative interactions ulti- lation of these paired altered specificity mutants indi- mately increases both the affinity and the DNA-binding cates that cooperative binding requires a direct protein– specificity of factors such as SNAP and TBP which, on protein contact, and suggests that this contact involves their own, display slow on and off rates and relatively K900 in SNAP190 and E7 in the Oct-1 POU domain. low DNA-binding specificity. Both SNAP and TBP con- K900 (Fig. 12, red triangle) and E7 (Fig. 12, blue triangle) tain built-in dampers of DNA binding. In the case of are, therefore, shown contacting each other in Figure 12. SNAP , the damper is deactivated by cooperative inter- In SNAP , then, the same region of the complex that is actions with Oct-1, which dramatically enhance the required for down-regulation of DNA binding is involved SNAP on rate (Mittal et al. 1996). In the case of TBP, the in a protein–protein interaction that mediates coopera- damper is deactivated by cooperative interactions with tive binding such that, in effect, cooperative binding in- SNAP (Mittal and Hernandez 1997). Because coopera- activates a SNAP built-in damper of DNA binding. tive interactions between Oct-1 and SNAP , and SNAP c c c GENES & DEVELOPMENT 2611 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez and TBP, only take place when the corresponding DNA- 50, 45, and 40 kD (Wang and Roeder 1998), whose iden- binding sites are all present, such interactions limit, in tities have not yet been reported. This relatively stable effect, the binding of these factors to promoter se- TFIIIC1/TFIIIC2 complex is referred to as human TFIIIC quences. (see Fig. 8). A TFIIIC1 activity purified by conventional chromatography, which may correspond to the four poly- peptides, binds cooperatively with both TFIIIC2 and a Stepwise assembly versus holoenzyme TFIIIC2/TFIIIA complex, and the footprint displayed by TFIIIC2 and the TFIIIC1 activity on a tRNA gene ex- Our understanding of how RNA polymerase III is re- tends over the A and B boxes (Oettel et al. 1997). cruited to specific promoters stems from in vitro studies In addition to the TFIIIC1 factor, the TFIIIC1 fraction in which transcription initiation complexes are as- contains an uncharacterized factor that specifically en- sembled by stepwise addition of various components. hances human U6 transcription and has been called Such studies have brought invaluable information about TFIIICU (Oettel et al. 1998) as well as an activity that the network of protein–DNA and protein–protein inter- footprints, on its own, over the terminator region of the actions that culminates in the specific recruitment of VAI gene and a tRNA gene (see Fig. 8; Wang and Roeder RNA polymerase III. In vivo, however, it is possible that 1996; Oettel et al. 1997, 1998). The latter activity is also many of the factors that mediate promoter recognition present in holo-TFIIIC but can be dissociated from the by RNA polymerase III are recruited together with the TFIIIC factor with 300 mM KCl (Wang et al. 2000). It has polymerase as part of a holoenzyme. Indeed, anti-epitope been purified by a combination of conventional and tag immunoprecipitates from a human cell line stably DNA affinity chromatography to near homogeneity, and synthesizing a tagged subunit of RNA polymerase III corresponds to four groups of NF1 isoforms (Wang et al. can direct transcription from type 2 promoters on their 2000). NF1 was initially identified as a cellular factor own and from type 1 promoters when combined with required for efficient initiation of Ad2 replication (Na- TFIIIA (Wang et al. 1997). Consistent with this observa- gata et al. 1982), and was later found to be involved in the tion, these immunoprecipitates contain the TFIIIB sub- expression of many cellular and viral genes. The purified units TBP and HsBrf1 as well as the TFIIIC2 subunits activity gives rise to a footprint over the two VAI termi- TFIIIC220 and TFIIIC110 as determined by immunoblot. nator regions that is identical to that observed with holo- Tandem immunoprecipitations as well as gel filtration TFIIIC, consistent with the idea that holo-TFIIIC con- analyses indicate that about 10% of the RNA polymer- tains, at a minimum, TFIIIC1, TFIIIC2, and NF1 poly- ase molecules in the immunoprecipitate is associated peptides (Fig. 8). The role of NF1 in RNA polymerase III with TFIIIB and/or TFIIIC components. However, these transcription is further addressed below. associations are reduced by an increase in the KCl con- centration from 100 mM to 150 mM, and are lost at 300 Termination and recycling: does the end help mM KCl. Nevertheless, these results raise the possibility the beginning? that in vivo, RNA polymerase III is recruited together with some of its accessory factors, in particular TFIIIB RNA polymerase III is unique among the eukaryotic and TFIIIC2 (Wang et al. 1997). RNA polymerases in recognizing a simple run of T resi- dues as a termination signal. In a linear template, muta- tion or deletion of the termination signal results in the Human TFIIIC1 production of run-off transcripts. However, aberrant ter- mination is not the only consequence of debilitating the The human TFIIIC fraction contains the B-box-binding terminator. Mutation of the run of T residues in the VAI TFIIIIC2 complex described above as well as the TFIIIC1 gene diminished the efficiency of single- and multiple- activity, which displays no strong DNA-binding activity round transcription in a HeLa cell extract (Wang and on its own but extends the TFIIIC2 footprint both up- Roeder 1996; Wang et al. 2000). Similarly, in an S. cer- stream on the A box and downstream to the end of the evisiae system in which RNA polymerase III was limit- gene (Yoshinaga et al. 1987). TFIIIC1 is apparently re- ing, deletion of the terminator affected multiple-round quired not only for transcription from type 1 and 2 pro- transcription, although in this case single-round tran- moters, but also for transcription from the type 3 gene- scription was not affected (Dieci and Sentenac 1996). external promoters (Yoon et al. 1995). As summarized in These results suggest that the run of T residues can con- Figure 8, this fraction contains a number of factors that tribute to the efficiency of initiation and reinitiation, have been implicated in RNA polymerase III transcrip- and therefore that it may play a role in RNA polymerase tion, many of which are still poorly characterized. Most recycling. In higher eukaryotes, a number of factors have of the TFIIIC1 activities can be purified as part of a holo- been implicated in efficient termination and RNA poly- TFIIIC complex by immunopurification of TFIIIC2 from merase III recycling, including the La protein, NF1 poly- a cell line expressing tagged TFIIIC220 (Wang and Roeder peptides, DNA topoisomerase I, and PC4. 1998). One of them, called TFIIIC1 (as opposed to the TFIIIC1 fraction), remains associated with TFIIIC2 after La protein further purification of the immunoprecipitated com- plex on a sucrose gradient and consists of at least four The first factor implicated in transcription termination polypeptides with apparent molecular masses of 70, and recycling was the La antigen (Gottlieb and Steitz 2612 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors 1989a,b). La binds to the poly-U tail at the end of RNA tive factors not present in the purified system (Wang et polymerase III transcripts (Stefano 1984), and could be al. 2000). Thus, NF1 can increase VAI transcription effi- demonstrated to stimulate transcription in a system in ciency in a crude extract, perhaps by facilitating tran- which transcription complexes were (1) preassembled on scription termination. immobilized templates; (2) allowed to undergo a single Depletion of NF1 reportedly also reduced transcription round of transcription in the presence of heparin and from the 5S RNA, a tRNA, and the human U6 snRNA genes, suggesting that NF1 polypeptides are generally re- sarcosyl, which resulted in stripping of RNA polymerase III; (3) washed; and (4) incubated with either fresh RNA quired for efficient RNA polymerase III transcription in polymerase alone or RNA polymerase together with La. crude extracts (Wang et al. 2000). It is not clear, however, The effect could be attributed to improved transcript re- how NF1 is recruited to other RNA polymerase III ter- lease and transcription reinitiation (Maraia 1996), and minators with no obvious NF1-binding sites close to the was abolished by phosphorylation of Ser 366 in La (Fan et run of T residues. Perhaps in these cases, protein–protein al. 1997). On the other hand, however, depletion of La interactions with TFIIIC2 (or, for genes such as the hu- from crude X. laevis extracts did not affect RNA poly- man U6 snRNA gene that do not recruit TFIIIC2, some merase III transcription (Lin-Marq and Clarkson 1998). other transcription factor) mediate NF1 recruitment. Thus, although La can clearly affect RNA polymerase III transcription in certain in vitro systems, it is not clear that the protein plays such a role in the cell. DNA topoisomerase I andPC4 Like NF1, both DNA topoisomerase I and PC4can sup- NF1 polypeptides press read-through transcripts in the dC-tailed template assay (Wang and Roeder 1998; Wang et al. 2000). How- Other factors implicated in transcription termination ever, these factors seem to differ from NF1 in that even and efficiency are the NF1 polypeptides in the TFIIIC1 though they enhance TFIIIC interactions in the down- fraction, which footprint over the VAI terminator region stream region of a tRNA gene and the VAI gene, they do (Wang et al. 2000). NF1 polypeptides form a family of not generate a footprint on their own, and the footprint proteins with multiple functions encoded by a large observed in the presence of TFIIIC does not appear to number of alternatively spliced RNAs derived from four extend over the termination region (Wang and Roeder different genes. Individual NF1 proteins can dimerize 1998; Wang et al. 2000). DNA topoisomerase I and PC4 with themselves and with other NF1 variants, but all are present in holo-TFIIIC, but unlike NF1, they are pres- variants have the same DNA-binding specificity and rec- ent only in trace amounts. Thus, DNA topoisomerase I ognize the consensus sequence 5-YTGGCANNNTGC and PC4seem less likely than NF1 to participate in RNA CAR-3. Sequences that closely match this consensus polymerase III transcription in vivo. are found in the two VAI terminators, immediately downstream of the run of T residues, and mutations in these NF1-binding sites resulted in the appearance of Conclusion read-through transcripts, although the effect was not as severe as with mutation of the run of T residues. Fur- The study of RNA polymerase III promoters has provided thermore, addition of NF1 together with the TFIIIC fac- perhaps the best illustration of how promoters with dif- tor improved the ratio of correctly terminated transcripts ferent structures rely differently on DNA–protein and over read-through transcripts derived from a dC-tailed protein–protein contacts to recruit, ultimately, the same template, in which RNA polymerase III initiates tran- RNA polymerase. Thus, TFIIIB can be recruited to a scription at the dC tail in the absence of transcription TATA box through direct binding to DNA, or to many initiation factors (Wang et al. 2000). Together, these re- DNA sequences through binding to TFIIIC. TFIIIC itself sults suggest an involvement of NF1 polypeptides in can bind directly to the B box of type 2 promoters or can transcription termination, although the interpretation of use the accessory factor TFIIIA to bind to type 1 promot- the dC-tailed template experiment is complicated by the ers. And in type 3 promoters, TFIIIB is recruited through fact that initiation at tailed templates tends to result in a combination of DNA contacts with the TATA box and protein contacts with SNAP the production of RNA–DNA hybrids, which are known . These promoters also il- to inhibit transcription termination by RNA polymerase lustrate how a transcription machinery has diversified III (Campbell and Setzer 1992). and expanded from S. cerevisiae with its relatively In addition to a qualitative effect on transcription ter- simple genome to higher eukaryotes with their much mination, NF1 polypeptides can affect transcription ef- more complex genomes to include new factors, such as ficiency. Thus, depletion of NF1 polypeptides from an SNAP , or multiple variations of a factor unique in yeast, extract severely reduced VAI transcription, and efficient such as HsBrf1 and HsBrf2. transcription could be restored by addition of either pu- RNA polymerase III transcription is tightly regulated; rified NF1 activity or recombinant CTF1, the largest it is controlled by growth conditions, during the cell NF1 variant derived from the NF1-C gene (Santoro et al. cycle, and by a number of viruses (for review, see Brown 1988). However, in a highly purified transcription sys- et al. 2000). These controls involve RNA polymerase III tem, the stimulatory effect of NF1 was reportedly not transcription factors, in particular TFIIIC2 and TFIIIB, as observed, suggesting that NF1 serves to counteract nega- well as, perhaps, RNA polymerase III itself. For example, GENES & DEVELOPMENT 2613 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez tion with TATA-binding protein. Cell 82: 857–867. the ratio of active TFIIIC2a versus inactive TFIIIC2b is Bai, L., Wang, Z., Yoon, J.-B., and Roeder, R.G. 1996. Cloning altered by the Ad2 E1A protein and the SV40 large T and characterization of the b subunit of human proximal antigen (Brown et al. 2000). TFIIIB activity is regulated sequence element-binding transcription factor and its in- by a number of factors including P53 and retinoblastoma volvement in transcription of small nuclear RNA genes by protein (Brown et al. 2000), as well as CK2, both in yeast RNA polymerases II and III. Mol. Cell. Biol. 16: 5419–5426. and human cells (Ghavidel and Schultz 2001; Johnston et Barberis, A., Müller, C.W., Harrison, S.C., and Ptashne, M. al. 2002). And in yeast, RNA polymerase III can be found 1993. Delineation of two functional regions of transcription associated with Maf1, a negative regulator of RNA poly- factor TFIIB. Proc. Natl. Acad. Sci. 90: 5628–5632. merase III transcription (Pluta et al. 2001). So far, recon- Bartholomew, B., Kassavetis, G.A., and Geiduschek, E.P. 1990. stitution of RNA polymerase III transcription with com- The subunit structure of Saccharomyces cerevisiae tran- pletely defined (recombinant) transcription factors has scription factor IIIC probed with a novel photocrosslinking reagent. EMBO J. 9: 2197–2205. been achieved only in the S. cerevisiae system with a ———. 1991. Two components of Saccharomyces cerevisiae promoter that can recruit TFIIIB directly through a transcription factor IIIB (TFIIIB) are stereospecifically lo- TATA box. An urgent task, which will allow a detailed cated upstream of a tRNA gene and interact with the second- understanding of the mechanisms by which RNA poly- largest subunit of TFIIIC. Mol. Cell. 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Willis, I.M. 2002. A universal nomenclature for subunits of the RNA polymerase III transcription initiation factor TFIIIB. Genes & Dev. 16: 1337–1338. Willis, I., Schmidt, P., and Soll, D. 1989. A selection for mutants 2620 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Laura Schramm and Nouria Hernandez Genes Dev. 2002, 16: Access the most recent version at doi:10.1101/gad.1018902 This article cites 224 articles, 128 of which can be accessed free at: References http://genesdev.cshlp.org/content/16/20/2593.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Cold Spring Harbor Laboratory Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes & Development Unpaywall

Recruitment of RNA polymerase III to its target promoters

Genes & DevelopmentOct 15, 2002

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Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Recruitment of RNA polymerase III to its target promoters Laura Schramm and Nouria Hernandez Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA A key step in retrieving the information stored in the (bp). This length limit is consistent with the elongation complex genomes of eukaryotes involves the identifica- properties of RNA polymerase III, which recognizes a tion of transcription units and, more specifically, the rec- simple run of T residues as a termination signal. The ognition of promoter sequences by RNA polymerase. In genes transcribed by RNA polymerase III encode RNA eukaryotes, the task of recognizing nuclear gene promot- molecules involved in fundamental metabolic processes, ers and then transcribing the genes is divided among specifically components of the protein synthesis appara- three highly related enzymes, RNA polymerases I, II, and tus and components of the splicing and tRNA processing III. Each of these RNA polymerases is dedicated to the apparatus, as well as RNAs of unknown function. The transcription of specific sets of genes, and each depends RNA polymerase III promoters are more varied in struc- on accessory factors, the so-called transcription factors, ture than the uniform RNA polymerase I promoters, and to recognize its cognate promoter sequences. yet not as diverse as the RNA polymerase II promoters. RNA polymerase I is unique among the nuclear RNA They have been divided into three main types, two of polymerases in transcribing only one set of genes, the which are gene-internal and generally TATA-less, and large, tandemly repeated, ribosomal RNA genes, and one of which is gene-external and contains a TATA box. thus in having to recognize a single promoter structure. Remarkably, we have a good, and in some cases a very RNA polymerase II transcribes the protein-coding genes detailed, understanding of how RNA polymerase III is (mRNA genes) as well as some small nuclear RNA recruited to each of these types of promoters. This pro- (snRNA) genes. The RNA polymerase II promoters can vides a paradigm of how the same enzyme can be re- be divided into a core region, defined as the minimal cruited to different promoter structures through differ- region capable of directing transcription in vitro, and a ent combinations of protein–DNA and protein–protein regulatory region. The regulatory regions are highly var- interactions. Here we summarize our present under- ied in structure, reflecting the highly varied synthesis standing of the various pathways leading to recruitment patterns of cellular proteins and the need for exquisite of RNA polymerase III. Other recent reviews on RNA and complex regulation of these patterns. The core pro- polymerase III transcription include those by Geidus- moters themselves come in different types that, in chek and Kassavetis (2001) and Huang and Maraia (2001). mRNA-encoding genes, can contain a TATA box, an ini- Reviews on the regulation of RNA polymerase III tran- tiator, a downstream promoter element, or various com- scription, which is not covered here, include those by binations thereof. The assembly of a functional RNA Ghavidel et al. (1999) and Brown et al. (2000). polymerase II transcription complex on a promoter con- sisting of just a TATA box has been extensively studied. Structure of RNA polymerase III promoters All the factors involved in the process have been identi- The three types of RNA polymerase III promoters are fied, and much is known about how these factors inter- called types 1–3. The first RNA polymerase III promoters act with DNA and with each other to recruit, eventually, characterized were those of the Xenopus laevis 5S RNA RNA polymerase II (for reviews, see Orphanides et al. gene (Bogenhagen et al. 1980; Sakonju et al. 1980), which 1996; Woychik and Hampsey 2002). How RNA polymer- encodes the small ribosomal RNA, the Adenovirus 2 ase II transcription complexes assemble on TATA-less (Ad2) VAI gene (Fowlkes and Shenk 1980), and various promoters is, however, not as well understood. tRNA genes from X. laevis and Drosophila melanogaster RNA polymerase III is dedicated to the transcription of (Galli et al. 1981; Hofstetter et al. 1981; Sharp et al. an eclectic collection of genes whose main common fea- 1981). The 5S promoter is the only example of a type 1 tures are that they encode structural or catalytic RNAs RNA polymerase III promoter, and the Ad2 VAI and and that they are, as a rule, shorter than 400 base pairs tRNA promoters are typical type 2 promoters. As shown in Figure 1, these promoters are intragenic. The X. laevis Corresponding author. 5S gene promoter consists of an A box, an intermediate E-MAIL [email protected]; FAX (516) 367-6801. element (IE), and a C box that is conserved in the 5S Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1018902. promoters of different species. Together, these elements GENES & DEVELOPMENT 16:2593–2620 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 2593 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez Figure 1. Different types of RNA polymerase III promoters. The type 1 promoter of the Xenopus laevis 5S RNA gene consists of an internal control region (ICR), which can be subdivided into A box (+50 to +60), intermediate element (IE, +67 to +72), and C box (+80 Leu to +90). The type 2 promoter of the X. laevis tRNA gene consists of an A box (+8 to +19) and a B box (+52 to +62). The type 3 promoter of the Homo sapiens U6 snRNA gene consists of a distal sequence element (DSE, −215 to −240) that enhances transcription and a core promoter composed of a proximal sequence element (PSE, −65 to −48) and a TATA box (−32 to −25). The Saccharomyces cerevisiae promoter is a hybrid promoter consisting of a TATA box (−30 to −23), an A box (+21 to +31), and a B box located downstream of the U6 coding region (from +234to +244relative to the start site of transcript ion). constitute the internal control region (ICR; Bogenhagen Clayton 1990), as well as in genes encoding RNAs of 1985; Pieler et al. 1985a,b, 1987). In the Saccharomyces unknown function. Their discovery came as a surprise cerevisiae 5S genes, only the C box is required for tran- because, unlike the then-characterized type 1 and 2 pro- scription (Challice and Segall 1989). moters, the type 3 core promoters turned out to be gene- The Ad2 VAI and most tRNA promoters consist of an external. As illustrated in Figure 1, they are located in A box and a B box (Galli et al. 1981; Hofstetter et al. the 5-flanking region of the gene and consist of a proxi- 1981; Sharp et al. 1981; Allison et al. 1983). These are mal sequence element (PSE), which also constitutes, on well conserved in tRNA genes from various species, its own, the core of RNA polymerase II snRNA promot- probably in part because they encode the tRNA D- and ers, and a TATA box located at a fixed distance down- T-loops, which are required for tRNA function. The stream of the PSE (Hernandez and Lucito 1988; Mattaj et spacing between the A- and B-boxes varies greatly, how- al. 1988; Kunkel and Pederson 1989; Lobo and Hernan- ever, in part to accommodate introns. The A-boxes of dez 1989). Strikingly, in the vertebrate snRNA promot- type 1 and 2 promoters are structurally related and are ers, RNA polymerase specificity can be switched from interchangeable in X. laevis (Ciliberto et al. 1983). How- RNA polymerase III to RNA polymerase II and vice versa ever, this apparently reflects a similarity in sequence by abrogation or generation of the TATA box (Lobo and rather than a conserved function, because, as detailed Hernandez 1989). Upstream of the PSE is an element below, the A-boxes of 5S and tRNA genes bind different referred to as the distal sequence element (DSE), which transcription factors (Braun et al. 1992a). activates transcription from the core promoter. The type 3 core promoters were identified originally in Although the presence of a TATA box is the hallmark mammalian U6 snRNA genes, which encode the U6 of type 3, gene-external, promoters, it is also found in the snRNA component of the spliceosome (Krol et al. 1987; 5-flanking regions of some genes with gene-internal pro- Das et al. 1988; Kunkel and Pederson 1988), and in the moter elements. Figure 1 shows an example of such a human 7SK gene (Murphy et al. 1986), whose RNA prod- hybrid promoter, namely the S. cerevisiae U6 snRNA uct has been recently implicated in the regulation of the promoter. It consists of an A box, a B box located at an CDK9/cyclin T complex (Nguyen et al. 2001; Yang et al. unusual position 120 bp downstream of the RNA coding 2001). They are also found in, for example, the H1 RNA region, and a TATA box located upstream of the tran- gene, which encodes the RNA component of human scription start site. All three of these promoter elements RNase P (Baer et al. 1989), and the gene encoding the are required for efficient transcription in vivo (Brow and RNA component of human RNase MRP (Topper and Guthrie 1990; Eschenlauer et al. 1993). Other examples 2594 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors include some A- and B-box-containing tRNA genes in ered, they were shown to require the B and C fractions, plants (Yukawa et al. 2000), yeast (Dieci et al. 2000), and or the B fraction and a D fraction eluted between 600 silkworm (Ouyang et al. 2000), in which TATA boxes mM and 1000 mM KCl from the phosphocellulose col- present in the 5-flanking region greatly contribute to umn (Lobo et al. 1991). Most of the activities in these transcription efficiency. More recently, an analysis in fractions required for RNA polymerase III transcription Schizosaccharomyces pombe has revealed that in this have now been characterized, both from yeast and hu- organism nearly all tRNA and 5S genes contain a TATA man cells. Figure 2 shows, in a highly simplified manner, box upstream of the transcription start site that is re- how these factors can assemble in an ordered fashion to quired for transcription (Hamada et al. 2001). Strikingly, recruit RNA polymerase III. The green arrows symbolize in vitro, artificial promoters consisting of just a TATA interactions of DNA-binding proteins with promoter el- box can direct RNA polymerase III transcription, indi- ements, the blue arrows protein–protein contacts among cating that under these circumstances, the TATA box various transcription factors, and the purple arrows pro- contains all necessary information to assemble an RNA tein–protein contacts between RNA polymerase III and polymerase III transcription initiation complex (Mitchell transcription factors. et al. 1992; Roberts et al. 1995; Wang and Stumph 1995; In type 2 promoters, the A and B boxes are recognized Whitehall et al. 1995; Huang et al. 1996). by a multisubunit complex in the C fraction called TFIIIC or TFIIIC2 (Lassar et al. 1983). This initial DNA– protein interaction then allows the recruitment of an The assembly pathways directed by the different activity in the B fraction called TFIIIB (Bieker et al. 1985; types of RNA polymerase III promoters converge Setzer and Brown 1985). TFIIIB is composed of three on recruitment of TFIIIB and RNA polymerase III polypeptides, one of which is the TATA-box-binding protein TBP. The binding of TFIIIB to the promoter in The characterization of RNA polymerase III transcrip- turn allows the recruitment of RNA polymerase III, tion factors started with the fractionation of a HeLa cell mainly through protein–protein interactions with extract over a phosphocellulose column into three frac- TFIIIB, although contacts with TFIIIC may also contrib- tions known as fractions A (the phosphocellulose 100 ute (Fig. 2A). In type 1 promoters, the ICR is recognized mM KCl flowthrough), B (a 100 mM–350 mM KCl step by the activity present in the A fraction, a zinc finger elution), and C (a 350 mM–600 mM KCl step elution), protein referred to as TFIIIA (Engelke et al. 1980; Sakonju and the observation that transcription from type 2 pro- moters required fractions B and C, whereas transcription et al. 1981). Formation of the TFIIIA–DNA complex then from type 1 promoters required the three fractions (Se- allows for the binding of TFIIIC (Lassar et al. 1983). gall et al. 1980). After the type 3 promoters were discov- Thus, TFIIIA can be viewed as a specificity factor that Figure 2. Different pathways for recruitment of TFIIIB and RNA polymerase III. The initiation complexes formed on type 2, 1, and 3 promoters, as well as on an artificial promoter consisting of just a TATA box, are shown. The green arrows symbolize interactions of DNA-binding proteins with promoter elements, the blue arrows protein–protein contacts among various transcription factors, and the purple arrows protein–protein contacts between RNA polymerase III and transcription factors. GENES & DEVELOPMENT 2595 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez alters the promoter-recognition properties of TFIIIC and unique system in which we know how different acces- targets it to the 5S promoter. After the binding of TFIIIC, sory factors combine to recruit, ultimately, a TFIIIB ac- the pathway to recruitment of the polymerase is similar tivity and RNA polymerase III. Below, we first describe to that in type 2 promoters, with the recruitment of briefly the subunit composition of RNA polymerase III. TFIIIB and RNA polymerase III (Fig. 2B). In type 3 pro- For a discussion of the likely three-dimensional struc- moters, the PSE is recognized by a multisubunit complex ture of RNA polymerase III, see Geiduschek and Kassa- variously called the PSE-binding protein (PBP), the PSE vetis (2001). We then describe the characterization of its transcription factor (PTF), or the snRNA activating pro- key transcription factor, TFIIIB, both yeast and human, tein complex (SNAP ), and the TATA box is recognized and we summarize our present understanding of how by the TBP component of a specialized TFIIIB-like activ- this factor bridges DNA and RNA polymerase III. We ity (Waldschmidt et al. 1991; Murphy et al. 1992; Sad- then summarize what is known about the various factors owski et al. 1993; Yoon et al. 1995; Schramm et al. 2000; that, in vivo, mediate the recruitment of TFIIIB on most, Teichmann et al. 2000). These DNA–protein interac- if not all, promoters, namely, TFIIIA, TFIIIC, and SNAP . tions are reinforced by protein–protein interactions be- We end with a description of some factors that have been tween SNAP and TBP (Mittal and Hernandez 1997; Ma implicated in termination and recycling of RNA poly- and Hernandez 2002). The binding of SNAP and the merase III. TFIIIB-like activity then lead to recruitment of RNA polymerase III (Sepehri Chong et al. 2001), probably RNA polymerase III through protein–protein contacts with the two DNA- bound factors, SNAP and the TFIIIB-like activity, al- RNA polymerase III is well defined in S. cerevisiae, con- though this has not yet been demonstrated (Fig. 2C). sisting of 17 subunits, as shown in Table 1. All the cor- Figure 2 also shows a recruitment pathway in which responding genes except for RPC37 have been disrupted TFIIIB is directly recruited to a TATA box without the and shown to be essential (for review, see Chedin et al. help of protein–protein contacts with either TFIIIC or 1998). Of the 17 subunits, 10 are unique to RNA poly- SNAP (Fig. 2D). This pathway can be observed in vitro merase III and are designated the C subunits, two are with S. cerevisiae TFIIIB, and, although it is not observed common to RNA polymerases I and III and are desig- in vivo, it reveals a profound aspect of RNA polymerase nated AC subunits, and five are common to the three III transcription, namely, that TFIIIB is sufficient for RNA polymerases and are designated ABC subunits. The RNA polymerase recruitment. TFIIIB was first identified common subunits have different names in RNA poly- as the key RNA polymerase III transcription factor by a merases I and II, as indicated in Table 1 for RNA poly- series of experiments in which S. cerevisiae TFIIIB was merase II. C160, C128, AC40, AC19, and ABC23 are evo- first recruited to either a 5S promoter through prior bind- lutionarily related to the core subunits of Escherichia ing of TFIIIA and TFIIIC, or a tRNA promoter through coli RNA polymerase, as indicated in parentheses in the prior binding of TFIIIC (Kassavetis et al. 1990). TFIIIA table. Of the C subunits, five, indicated in bold in Table and/or TFIIIC were then stripped from the DNA by treat- 1, are specific to RNA polymerase III. ment with heparin or high concentrations of salt. Under Human RNA polymerase III has been purified both by these conditions, functional TFIIIA and TFIIIC were re- conventional chromatography (Wang and Roeder 1996) leased from the templates, but remarkably, TFIIIB re- and from cell lines expressing tagged Homo sapiens (Hs) mained bound to the DNA, generating the same foot- RPC4/RPC53/BN51 (Wang and Roeder 1997), but until print upstream of the transcription start site as it did in recently, only five of its subunits had been characterized: the presence of TFIIIA and/or TFIIIC (Kassavetis et al. HsRPC4/RPC53 (Ittmann et al. 1993; Jackson et al. 1989, 1990). These stripped templates were able to sup- 1995), HsRPC1/RPC155 (Sepehri and Hernandez 1997), port several rounds of properly initiated RNA polymer- HsRPC3/RPC62, HsRPC6/RPC39, and HsRPC7/RPC32 ase III transcription. This suggested that, at least in (Wang and Roeder 1997). Human RNA polymerase III yeast, TFIIIB was sufficient to recruit RNA polymerase has now been purified from a stable cell line expressing III and direct several rounds of transcription, and there- a doubly tagged HsRPC4subunit, and its subunits have fore that the main function of TFIIIA and TFIIIC was to been identified by mass spectrometry (Hu et al. 2002). recruit TFIIIB to the DNA (Kassavetis et al. 1990). With This analysis has resulted in the identification of or- the observation that just a TATA box could direct RNA thologs of all of the yeast RNA polymerase III subunits polymerase III transcription in vitro and with the avail- except for ABC10, which was not detected probably ability of recombinant TFIIIB, it then became possible to because of its small size (7 kD). The newly described confirm that a TATA box could direct several rounds of human subunits were named according to the guide RNA polymerase III transcription with just recombinant shown in the fourth column of Table 1, in which the TFIIIB and highly purified RNA polymerase III (Kassa- yeast C, AC, and ABC subunits were numbered sepa- vetis et al. 1995; Rüth et al. 1996). Thus, TFIIIA, TFIIIC, rately in order of decreasing apparent molecular weight. and SNAP can be viewed as recruitment factors whose Such a nomenclature would provide the same name for main function is to recruit TFIIIB to promoters of various orthologs from different species, as shown in the fourth structures, which then allows the recruitment of RNA and sixth column in Table 1. polymerase III. The characterization of human RPC8 and RPC9 The different RNA polymerase III promoters offer a brought an unexpected result. BLAST searches revealed 2596 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Table 1. Subunits of Saccharomyces cerevisiae and Homo sapiens RNA polymerase III % amino acid Corresponding identities between S. cerevisiae S. cerevisiae H. sapiens and RNA Pol III MW Accession Guide for RNA Pol II H. sapiens RNA MW S. cerevisiae subunits (kD) number nomenclature subunits RNA Pol III subunits (kD) Accession number Pol III subunits C160 (-like) 162.1 P04051 ScRPC1 RPB1 HsRPC1/RPC155 155.6 AAB86536 50% (1356/1391) C128 (-like) 129.3 AAB59324ScRPC2 RPB2 HsRPC2 127.6 AY092084 63%(1115/1133) C82 73.6 CAA45072 ScRPC3 HsRPC3/RPC62 60.5 NP_006459/XP_034604 22% (163/534) C53 46.6 P25441 ScRPC4 HsRPC4/RPC53 44.4 AY092086 28% (134/398) C37 32.1 NP_012950 ScRPC5 HsRPC5 79.8 AY092085 26% (160/708) C34 36.1 P32910 ScRPC6 HsRPC6/RPC39 35.6 NP_006457/XP_009639 26% (216/316) C31 27.7 P17890 ScRPC7 HsRPC7/RPC32 25.9 AAB63676/XP_036456 35% (44/223) C25 24.3 P35718 ScRPC8 RPB7 HsRPC8 22.9 AY092087 42% (201/204) C17 18.6 P47076 ScRPC9 RPB4 HsRPC9/CGRP-RC 16.8 AAC25992 30% (122/148) C11 12.5AAD12060ScRPC10 RPB9 HsRPC10/RPC11 12.3NP_05739452%(108/108 ) AC40 (-like) 37.6 P07703 ScRPAC1 RPB3 HsRPAC1/RPA5,RPA39 38.6 NP_004866 47% (287/342) AC19 (-like) 16.1 P28000 ScRPAC2 RPB11 HsRPAC2/RPA9,RPA16 15.2 NP_057056 45% (119/133) ABC27 25.1 P20434 ScRPABC1 RPB5 HsRPABC1/RPB5,RPB25 24.6 P19388 42% (207/210) ABC23 (-like) 17.9 AAA34989 ScRPABC2 RPB6 HsRPABC2/RPB6,RPB14.4 14.5 P41584 72% (83/127) ABC14.5 16.5 CAA37383 ScRPABC3 RPB8 HsRPABC3/RPB8,RPB17 17.1 P52434 35% (147/150) ABC10 7.7 AAA64417 ScRPABC4 RPB12 HsRPABC4/RPB7.0 7.0 P53803 52% (42/58) ABC10 8.2 P22139 ScRPABC5 RPB10 HsRPABC5/RPB10,RPB7.6 7.6 P52436 73% (67/67) Subunits in bold do not have paralogues in RNA polymerases I and II; those in bold and underlined form a complex separable from the rest of the enzyme. Subunits corresponding to the E. coli , , , and  subunits are indicated. ABC27 and RPB5, ABC23 and RPB6, ABC14.5 and RPB8, ABC10 and RPB12, ABC10 and RPB10 designate in each case the same protein. For HsRPC62, HsRPC39, and HsRPC32, the sequence under the first accession number (Wang and Roeder 1997) differs in several positions from both the sequences deposited by NCBI (second accession number) and genomic sequences. The first number in the parentheses indicates the length of the region of similarity; the second number indicates the total length of the human protein. Reprinted from Hu et al. (2002). that HsRPC8 is related to the RNA polymerase II sub- in transcription initiation, but in this case both subunits unit RPB7, as noted earlier for the S. cerevisiae HsRPC8 are essential for yeast cell viability, perhaps because ortholog C25 (Sadhale and Woychik 1994). In addition, most RNA polymerase III genes encode components es- however, HsRPC9 is related to RPB4, and like RPB4 and sential for cell metabolism. RPB7, which associate with each other and form a dimer The human RNA polymerase III subunits are in gen- detachable from the rest of RNA polymerase II (Edwards eral quite similar to their yeast counterparts with the et al. 1991; Khazak et al. 1998), HsRPC8 and HsRPC9 notable exception of the subunits with no paralogs in associate with each other (Hu et al. 2002). This strongly RNA polymerase II (Jackson et al. 1995; Wang and suggests that HsRPC8 and HsRPC9 are paralogs of RPB7 Roeder 1997; Hu et al. 2002). For example, the human and RPB4, as indicated in Table 1, and that the corre- ortholog of yeast C37 is an 80-kD protein, HsRPC5, sponding S. cerevisiae RNA polymerase III subunits C25 whose similarity to the yeast protein is confined to its and C17 can similarly associate with each other. N-terminal fourth, which shows 26% identity with C37 RPB7, but not RPB4, is essential for yeast cell viability (Hu et al. 2002). Nevertheless, like yeast C37, which (Woychik and Young 1989; McKune et al. 1993). RPB4 associates with the yeast C53 subunit (Flores et al. 1999), is, however, essential for cellular responses to stress HsRPC5 associates with HsRPC4/RPC53, the human or- (Choder and Young 1993) and thus in vivo, the require- tholog of yeast C53, and this association is through the ment for the RPB4subunit may be promoter-specific. HsRPC5 and HsRPC4/RPC53 domains conserved in The RPB4/RPB7 complex is thought to stabilize the open their yeast counterparts (Hu et al. 2002). Interestingly, at promoter complex and perhaps the early transcribing least some of the subunits with no paralogs in RNA poly- complex prior to promoter escape by binding to nascent merase II seem to be involved in promoter recognition. RNA or to single-stranded DNA in the transcription The C82, C34, and C31 subunits (bold and underlined in bubble (Orlicky et al. 2001; Todone et al. 2001). The S. Table 1) dissociate from a yeast enzyme carrying a mu- cerevisiae RNA polymerase III paralogs of RBP7 and tation within the zinc finger domain of the largest sub- RPB4, C25 and C17, are both essential for viability in unit, and each associates with the two others in a yeast yeast (Sadhale and Woychik 1994; Ferri et al. 2000). Two- two-hybrid assay, suggesting that these three subunits hybrid and coimmunoprecipitation experiments indicate form a subcomplex detachable from the rest of the en- that C17 interacts with the transcription initiation fac- zyme (Werner et al. 1992, 1993). In the human enzyme, tor Brf1 and with the RNA polymerase III C31 subunit such a subcomplex could be demonstrated directly by (Ferri et al. 2000), which, as described below, is itself sucrose gradient centrifugation under partially denatur- required for transcription initiation (Werner et al. 1992, ing conditions and by reconstitution of the subcomplex 1993; Wang and Roeder 1997). Thus, the RNA polymer- from recombinant subunits (Wang and Roeder 1997). ase III paralogs of RPB4and RPB7 may also be involved The subunits in the subcomplex are not required for ef- GENES & DEVELOPMENT 2597 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ficient elongation and termination, but are required for ratowski and Zhou 1992). This ability to suppress muta- specific initiation (Thuillier et al. 1995; Brun et al. 1997; tions in TBP suggested that Brf1 might be associated Wang and Roeder 1997). Consistent with this observa- with TBP, and thus that TBP might be part of the TFIIIB tion and as detailed further below, the C34subunit and activity. Indeed, TBP was shown to be part of both the its human counterpart interact directly with TFIIIB sub- yeast and mammalian TFIIIB activity by biochemical units (Werner et al. 1993; Khoo et al. 1994; Wang and methods (Margottin et al. 1991; Huet and Sentenac 1992; Roeder 1997). Kassavetis et al. 1992; Lobo et al. 1992; Taggart et al. 1992; White and Jackson 1992; Chiang et al. 1993; Mey- ers and Sharp 1993), and to constitute a previously un- recognized, Brf1-associated, component of the B activity Composition of TFIIIB (Kassavetis et al. 1992). The cloning of the gene encoding TBP is requiredfor transcription by RNA polymerase yeast B (B, Kassavetis et al. 1995; TFIIIB90, Roberts et III both in S. cerevisiae andhuman cells al. 1996; TFC7p, Rüth et al. 1996), now referred to as Bdp1 (B double prime 1, Willis 2002), then completed the Although the presence of an RNA polymerase III tran- characterization of S. cerevisiae TFIIIB. scription activity in the phosphocellulose B fraction was recognized in the early 1980s, the composition of this activity remained a mystery for the next 10 years. By the Identification of human Brf1 and Brf2 late 1980s, however, the concept that the TATA-box- In S. cerevisiae, all RNA polymerase III promoters re- binding protein TBP was a factor uniquely dedicated to cruit the same TFIIIB factor (Joazeiro et al. 1994). In transcription by RNA polymerase II began to change higher eukaryotes, however, the situation is more com- with the finding that an essential element of the U6 plex, consistent with the need to transcribe much more promoter was an A/T-rich region, that is, a potential complex genomes. Thus, the initial characterization of binding site for TBP. Biochemical fractionation and re- mammalian TFIIIB not only indicated that TBP was part constitution experiments then identified TBP as a factor of the activity (Lobo et al. 1992; Taggart et al. 1992; required for transcription of both the yeast and human White and Jackson 1992), but also that type 1 and 2 pro- U6 snRNA genes (Lobo et al. 1991; Margottin et al. 1991; moters used different components in the TFIIIB fraction Simmen et al. 1991), whose binding to wild-type and than type 3 promoters. Type 1 and 2 promoters were mutant U6 TATA boxes correlated with transcription shown to require a TBP-containing complex (Lobo et al. activity (Lobo et al. 1991). These findings established 1992; Teichmann and Seifart 1995) consisting of TBP and that TATA boxes are part of at least some RNA poly- a homolog of yeast Brf1 (Wang and Roeder 1995; Mital et merase III promoters, and that they act by recruiting al. 1996) referred to as HsBrf1 (Homo sapiens Brf1). TBP. They also raised the possibility that TBP might be Depletion of extracts with antibodies directed against required for RNA polymerase III transcription in general. the C-terminal half of HsBrf1 debilitated transcription Indeed, in vitro competition experiments with TATA- from the type 2 VAI promoter, as expected, but had no containing oligonucleotides then indicated that a TATA- effect on transcription from the type 3 human U6 box-binding factor was required for transcription of the snRNA promoter (Mital et al. 1996; Henry et al. 1998a). VAI and tRNA genes (White et al. 1992), and inactivation On the other hand, depletion of extracts with antibodies of TBP in yeast was shown to lead to defects in transcrip- raised against full-length HsBrf1 or against a peptide de- tion by all three RNA polymerases (Cormack and Struhl rived from the N-terminal portion of the protein inhib- 1992; Schultz et al. 1992). The remaining question was ited transcription from all types of RNA polymerase III how to place TBP in what was then known about RNA promoters, although only transcription from type 1 and 2 polymerase III transcription factors. promoters could be reconstituted by addition of recom- binant HsBrf1 (Wang and Roeder 1995; Schramm et al. 2000). These observations suggested that type 3 promot- Identification of S. cerevisiae Brf1 andBdp1 ers use a protein related to Brf1 in its N-terminal but not Yeast TFIIIB had been shown to consist of two chromato- its C-terminal region, and led to the characterization of a graphically separable activities, named B and B, which new protein, originally called BRFU (Schramm et al. contained polypeptides of 70 and 90 kD, respectively, 2000) or TFIIIB50 (Teichmann et al. 2000), and now re- that could be cross-linked to the DNA (Bartholomew et ferred to as HsBrf2 (Willis 2002). Thus, S. cerevisiae Brf1 al. 1991; Kassavetis et al. 1991). A major step in the com- has at least two homologs in human cells, HsBrf1 and plete characterization of TFIIIB came with the cloning of HsBrf2. the gene encoding the 70-kD polypeptide, now referred Figure 3 shows the structure of TFIIB and various Brf to as Brf1 (TFIIB-related factor 1; for a description of a proteins. H. sapiens and S. cerevisiae (Sc) Brf1 as well as universal nomenclature of TFIIIB components, see Willis HsBrf2 contain, like TFIIB, an N-terminal zinc-binding 2002). The gene was cloned as a suppressor of a tRNA domain (green box) and a “core domain” consisting of gene A-box mutation and called PCF4 (López-De-León et two imperfect repeats (blue box). In addition, the Brf1 al. 1992). It was also cloned, however, as an allele-spe- and Brf2 proteins contain C-terminal domains absent in cific high-copy suppressor of certain mutations in TBP TFIIB. Within the C-terminal segment of Brf1, three re- and called BRF1 (Colbert and Hahn 1992) or TDS4 (Bu- gions, designated regions I, II, and III, are conserved in 2598 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Figure 3. TFIIB, Brf1, and Brf2 form a family of related transcription factors. The location of the structured zinc ribbon as modeled in ScTFIIB and ScBrf1 (Hahn and Roberts 2000) based on the NMR structure of PfTFIIB (Zhu et al. 1996) and that of the corresponding region in HsBrf1 and HsBrf2 is indicated in green. The location of the structured core domain of TFIIB (Bagby et al. 1995; Nikolov et al. 1995) and that of the corresponding regions in the other proteins is indicated in blue. The percentages below the sequences indicate percent identities between HsBrf2 and HsTFIIB, ScTFIIB, ScBrf1, and HsBrf1 within the region of highest conservation (bracketed by the stippled lines) in pairwise alignments performed with BLAST. The purple boxes in the C-terminal regions of ScBrf1 and HsBrf1 indicate conserved regions I, II, and III (Mital et al. 1996). In HsBrf1_v2, the blue region is identical to the corresponding HsBrf1 region. the yeasts Candida albicans, Kluyveromyces lactis, S. well as the C-terminal region present in Brf1. Although pombe, and S. cerevisiae (Khoo et al. 1994). Regions II HsBrf1 is not involved in human U6 transcription, and III are also conserved in the human Brf1 protein (Mi- HsBrf1_v2 has been implicated in U6 transcription be- tal et al. 1996; Andrau et al. 1999). Consistent with the cause when antibodies recognizing all HsBrf1 variants antibody depletion data, the C-terminal domain of were used to deplete extracts, U6 transcription was lost HsBrf2 shows very little, if any, homology with Brf1. and could be specifically restored by addition of material HsBrf2 was isolated through a database search for pro- immunopurified from cells expressing tagged HsBrf1_v2 teins related to TFIIB and to the TFIIB-related segment of (McCulloch et al. 2000). It will be necessary to define the Brf1 (Schramm et al. 2000), as well as through biochemi- composition of this immunopurified fraction to confirm cal purification of a complex, consisting of HsBrf2 and the role of HsBrf1_v2 in U6 transcription. four associated proteins, required for transcription from type 3 promoters (Teichmann et al. 2000). It is clear that Identification of human Bdp1 HsBrf2 itself is specifically required for transcription from type 3, but not types 1 and 2, promoters, but the Figure 4shows the structure of S. cerevisiae Bdp1. It exact role of the HsBrf2-associated factors remains to be contains a domain related to a Myb repeat, identified in determined. Although in one case, U6 transcription in the SWI–SNF and ADA complexes, the transcriptional HsBrf2-depleted extracts could be restored only by addi- corepressor N-Cor, and yeast TFIIIB Bdp1, and therefore tion of the HsBrf2-containing complex immunopurified referred to as the SANT domain (Aasland et al. 1996). from HeLa cells expressing tagged HsBrf2 (Teichmann et The SANT domain is absolutely required for TFIIIC-de- al. 2000), in another case it could be restored by addition pendent (but not TFIIIC-independent, see below) RNA of just HsBrf2 synthesized in E. coli (Schramm et al. polymerase III transcription (Kumar et al. 1997). In addi- 2000). This last observation suggests that the HsBrf2- tion, a region upstream of the SANT domain (indicated associated polypeptides may not be absolutely required in orange in Fig. 4) is required for transcription from for U6 transcription but may contribute to the efficiency linear, but not supercoiled, templates (Kassavetis et al. of the reaction. 1998a). Figure 3 also illustrates the structure of HsBrf1_v2 Human Bdp1 cDNAs were isolated through a combi- (originally named BRF2), a factor encoded by one of at nation of database searches for sequences similar to the least four alternatively spliced BRF1 pre-mRNAs (Mc- yeast Bdp1 SANT domain and library screening Culloch et al. 2000). HsBrf1_v2 lacks the zinc finger do- (Schramm et al. 2000). The structure of the protein en- main and the first repeat that are present in Brf1 and coded by one of these cDNAs (HsBdp1) is shown in Fig- conserved in the other proteins of the TFIIB family, as ure 4. It is highly related to the yeast protein within the GENES & DEVELOPMENT 2599 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez Figure 4. Comparison of the ScBdp1 and HsBdp1 polypeptides. The proteins contain a conserved SANT domain (brown box). The regions upstream and downstream of the SANT domain are also quite conserved, especially a segment upstream of the SANT domain (indicated in orange) that is required for transcription from linear, but not supercoiled, templates. The percentages indicate amino acid identities between ScBdp1 and HsBdp1 in the regions bracketed by dotted lines. HsBdp1, HsBdp1_v2, and HsBdp1_v3 are identical in the colored regions. The repeats extend from amino acids 822 to 1338. HsBdp1 and HsBdp1_v2 diverge after amino acid 1353. HsBdp1 and HsBdp1_v3 diverge after amino acid 684. SANT domain (43% identity) as well as both immedi- The characterization of human TFIIIB has revealed ately upstream, in a region that encompasses the seg- that unlike in S. cerevisiae, where type 3 promoters ap- ment required for transcription from linear DNA tem- parently do not exist and one form of TFIIIB serves all plates (21% identity), and downstream (17% identity). RNA polymerase III promoters (Joazeiro et al. 1994), Outside of these regions, the two proteins are not con- there are at least two forms of TFIIIB in human cells. As served, and the human protein differs from the yeast pro- shown in Figure 5, one of them consists of HsTBP, tein by a striking C-terminal extension containing a HsBrf1, and HsBdp1 and is used by type 2 (and probably number of repeats with potential phosphorylation sites. type 1) promoters. The other consists of HsTBP, HsBrf2, A number of alternatively spliced BDP1 cDNAs have and HsBdp1, and is used by type 3 promoters. Future been isolated (Kelter et al. 2000; Schramm et al. 2000). work may reveal that different spliced variants of Bdp1 Two of these encode strikingly different proteins, which are recruited to different RNA polymerase III promoters are also shown in Figure 4. The longest protein (labeled in vivo. Furthermore, in D. melanogaster cells, the TBP HsBdp1_v2 in the figure) is identical to Bdp1 except that in TFIIIB is replaced by a TBP-related factor called TRF1 the last few amino acids are replaced by a 901-amino- (Takada et al. 2000). Thus, there may be a wide range of acid extension, giving a protein of 2254amino acids. TFIIIB activities in different species containing variants Another cDNA encodes a 725-amino-acid protein of each of the three TFIIIB components. (Bdp1_v3), which contains Bdp1 sequences up to amino In S. cerevisiae, H. sapiens, and D. melanogaster, Brf1 is tightly associated with TBP or TRF1 in solution, as acid 684, followed by a divergent 47-amino-acid exten- sion (Kelter et al. 2000). symbolized by red bars in Figure 5. On the other hand, Which of the alternatively spliced forms of human Bdp1 is weakly associated with the TBP–Brf1 complex in Bdp1 are involved in RNA polymerase III transcription in S. cerevisiae, and very weakly, if at all, in human cells vivo is not clear at present. Depletions of extracts with (Kassavetis et al. 1991; Wang and Roeder 1995; Mital et antibodies directed against regions both upstream and al. 1996; Schramm et al. 2000). Indeed, an association downstream of the SANT domain within the N-terminal between HsBdp1 and HsTBP can only be detected in GST half of human Bdp1 (Schramm et al. 2000), as well as pull-downs (blue bars in Fig. 5; Cabart and Murphy against the repeat region (L. Schramm and N. Hernandez, 2002). Similarly, although HsBrf2 can be shown to asso- unpubl.), debilitate transcription from both type 2 and 3 ciate with HsTBP in GST pull-downs (Cabart and Mur- promoters in vitro, and transcription can be restored by phy 2001, 2002), it is not strongly associated with TBP in addition of recombinant human Bdp1, either full-length HeLa cell extracts (Schramm et al. 2000). Thus, the or truncated downstream of the SANT domain. This sug- TFIIIB components do not always form a stable complex gests that HsBdp1 is generally required for RNA poly- off the DNA. merase III transcription, and that the C-terminal repeats are not required for basal in vitro transcription from na- Functions of TFIIIB ked DNA templates. However, the functional protein present in HeLa cell extracts probably contains the re- In RNA polymerase II transcription, the opening of the peat region, because it can be depleted by antibodies di- transcription bubble that occurs after recruitment of the rected against this region. Perhaps the repeat region per- polymerase is dependent on TFIIE and an ATP-depen- forms a regulatory role not scored in the in vitro tran- dent helicase activity of TFIIH (Holstege et al. 1996; scription assay. Tirode et al. 1999). In contrast, in RNA polymerase III 2600 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors Figure 5. Promoter-selective TFIIIB activities. The TFIIIB components required by different classes of promoters in Homo sapiens and Drosophila melanogaster are depicted. Strong (red bars) and weak (blue bars) direct protein–protein associations in solution are indicated. Stippled lines indicate that a direct protein–protein contact has not been demonstrated. DmBdp1 has not been characterized, but a candidate gene has been identified (Schramm et al. 2000). allows it to bind to the mutated TATA box (Strubin and transcription, the opening of the transcription bubble oc- curs in an ATP-independent manner after recruitment of Struhl 1992; Whitehall et al. 1995). The TBP–TATA-box RNA polymerase III by TFIIIB (Kassavetis et al. 1990, complex is then recognized by Brf1 or, in the case of the 1992). The availability, in yeast, of both recombinant human U6 promoter, by Brf2. The similarity of both Brf1 TFIIIB and a transcription system independent of TFIIIC, and Brf2 to TFIIB is very striking, and immediately sug- that is, a system in which a TATA box can recruit TFIIIB gests that the conserved domains of these proteins may directly, has allowed detailed analyses of the functions of perform equivalent functions during assembly of RNA the TFIIIB subunits. These studies have given a detailed polymerase II and III transcription initiation complexes. picture of how TFIIIB recognizes the TATA box and how The reality, however, is more complex. In TFIIB, the core it recruits RNA polymerase III. They have also revealed domain is sufficient for association with the TATA-box– that, remarkably, TFIIIB not only functions to recruit TBP complex. However, recruitment of RNA polymer- RNA polymerase III but also participates in opening of ase II and TFIIF to the TATA-box–TBP–TFIIB complex the transcription bubble. requires the TFIIB zinc-binding domain (Barberis et al. 1993; Ha et al. 1993; Hisatake et al. 1993; Yamashita et al. 1993; Pardee et al. 1998). HsBrf2 resembles TFIIB in that it recognizes the Binding of TFIIIB to the TATA box TATA-box/TBP complex through its TFIIB-related core In promoters consisting of just a TATA box, S. cerevisiae domain (Cabart and Murphy 2001). In contrast, for S. TFIIIB binds to the DNA through recognition of the cerevisiae Brf1, the task of recognizing the TBP–TATA- TATA box by its TBP subunit. Indeed, a mutation in the box complex is performed by two regions of the protein, TATA box that debilitates RNA polymerase III tran- the TFIIB-related N-terminal half as well as the Brf1- scription can be compensated by a mutation in TBP that specific C-terminal half, with the latter playing the ma- alters the DNA-binding specificity of the protein and jor role. Thus, as summarized in Figure 6, a truncated Figure 6. Functional domains of Saccharomyces cerevisiae Brf1. S. cerevisiae Brf1 is depicted, with the locations of the zinc domain, direct repeats in the core, and conserved regions I, II, and III. The brackets below indicate regions of the proteins sufficient for association with the TBP–TATA-box complex (Kassavetis et al. 1998b), TBP alone (Khoo et al. 1994), and the C34 (Khoo et al. 1994; Andrau et al. 1999) and C17 (Ferri et al. 2000) subunits of RNA polymerase III. The black boxes indicate regions where mutations or deletions have a strong negative effect on the associations. The stippled line indicates an association detected only by UV cross- linking. The upstream boundary of the Brf1 region sufficient for interaction with C17 is not precisely defined. GENES & DEVELOPMENT 2601 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ScBrf1 protein retaining just the zinc-binding domain TATA-box–TBP–HsBrf2 complex (Cabart and Murphy and the core associates only very weakly with a TATA- 2002). box–TBP complex. Indeed, the association is so weak that it is only detected by methods such as photochemi- cal cross-linking (Kassavetis et al. 1997, 1998b; Colbert RNA polymerase recruitment by TFIIIB et al. 1998). This weak association appears to involve a Figure 7 shows the known protein–protein contacts be- TBP surface that overlaps or lies near the TFIIB-interact- tween TFIIIB and RNA polymerase III subunits with ar- ing surface in the “stirrup” of the second TBP repeat, rows for contacts identified with human (solid) or yeast because a triple-amino-acid change in TBP that disrupts (hatched) subunits, respectively. Eight RNA polymerase the TFIIB interaction suppresses cross-linking of the N- III subunits can be cross-linked to DNA in a transcrip- terminal half of ScBrf1 to DNA and thus probably asso- tion initiation complex (Bartholomew et al. 1993). Of ciation with the TBP–TATA-box complex (Kassavetis et these, C34, which is part of the three-subunit subcom- al. 1998b). On the other hand, a 110-amino-acid region plex that is required for transcription initiation (Werner encompassing conserved region II within the C-terminal et al. 1993; Wang and Roeder 1997), maps the furthest half of the protein is sufficient for stable association with upstream and can be localized between positions −17 and a TATA-box–TBP complex as well as for recruitment of +6 relative to the transcription start site, in close prox- ScBdp1. Moreover, the hydroxyl radical footprint ob- imity to TFIIIB (Bartholomew et al. 1993). ScBrf1 inter- served with just the C-terminal domain of ScBrf1 is iden- acts in vivo and in vitro with C34, and human Brf1 as- tical to that observed with the full-length protein (Col- sociates with the human homolog of C34, HsRPC39, in bert et al. 1998). Thus, despite the strong conservation of vitro (Werner et al. 1993; Khoo et al. 1994; Wang and the core domains in TFIIB and Brf1, it appears that in Roeder 1997). As shown in Figure 6, ScBrf1 appears to ScBrf1, the function of recognizing the TBP–TATA-box contact C34through three regions: regions II and III complex has been largely transferred to the C-terminal within the Brf1-specific C-terminal domain (Andrau et half of the protein and in particular to conserved region al. 1999), and another region, identified by GST pull- II. This region of ScBrf1 binds the opposite face of the down assays, located within the core region in the TFIIB- TBP–TATA-box complex from TFIIB and recognizes a related N-terminal half of the protein (Khoo et al. 1994). TBP surface that overlaps that recognized by TFIIA (Col- ScBrf1 also contacts the recently identified RNA poly- bert et al. 1998; Kassavetis et al. 1998b; Shen et al. 1998; merase III subunit C17 through the C-terminal half of its for models of the structure of the TBP–DNA–ScBrf1 core region (Ferri et al. 2000). Notably, unlike the zinc- complex, see Colbert et al. 1998; Geiduschek and Kassa- binding domain of TFIIB, the zinc-binding domain of vetis 2001). It will be important to contrast HsBrf1 and ScBrf1 is not required for RNA polymerase recruitment HsBrf2 with ScBrf1 to determine how these TBP-associa- (Kassavetis et al. 1997; Hahn and Roberts 2000). How tion activities have been conserved among the human HsBrf2 contacts RNA polymerase III is not known. It Brf1 and Brf2 proteins. will be highly interesting to determine further which ScBdp1 can associate with a preassembled TBP– parts of the protein are required for assembly with TBP ScBrf1–TATA-box complex but not with a complex lack- and SNAP onto the human U6 promoter and for recruit- ing ScBrf1, and this confers on the yeast TFIIIB–DNA ment of RNA polymerase III. Contacts between Bdp1 complex its striking resistance to salt and heparin. and RNA polymerase III subunits have not been de- ScBdp1 contacts not only ScBrf1 but also TBP, because at scribed, but as shown in Figure 7, human TBP associates least one mutation in yeast TBP prevents association of ScBdp1 without affecting association of ScBrf1 (Colbert et al. 1998). ScBdp1 also contacts DNA because its as- sembly onto the TATA-box–TBP–ScBrf1 complex both requires DNA, and extends the DNA footprint, upstream of the TATA box (Colbert et al. 1998; Shah et al. 1999). Moreover, ScBdp1 can be cross-linked to the DNA at sites upstream of the TATA box (Shah et al. 1999). The binding of ScBdp1 to the TBP–ScBrf1–TATA-box com- plex induces a bend in the DNA between the TATA box and the transcription start site, which is in phase with the bend imposed by TBP on the TATA box (Leveillard et al. 1991; Braun et al. 1992b; Grove et al. 1999). This bending of the DNA has been postulated to contribute to the ScBdp1-dependent stabilization of the TFIIIIB–DNA complex by helping impede sliding of the DNA out of the complex (Grove et al. 1999), a hypothesis consistent Figure 7. Protein–protein contacts between TFIIIB compo- with thermodynamic and kinetic data indicating nents and RNA polymerase III subunits. The solid arrows rep- ScBdp1-dependent kinetic trapping of the DNA (Cloutier resent contacts identified with human subunits, the stippled et al. 2001). In the human system, HsBdp1 has been arrows depict contacts identified with Saccharomyces cerevi- shown to assemble, albeit inefficiently, on a preformed siae subunits. 2602 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors with the HsRPC39 RNA polymerase III subunit in vitro Recruitment factors: TFIIIA (Wang and Roeder 1997). In an in vitro system in which TFIIIB can be recruited directly to a TATA box, TFIIIB on its own is sufficient to recruit RNA polymerase III. In natural RNA polymerase Post-RNA polymerase III recruitment roles for Brf1 III promoters, however, TFIIIB is recruited to the DNA in andBdp1 large part through protein–protein contacts with pro- moter-bound recruitment factors, specifically TFIIIC or In in vitro transcription assays with supercoiled tem- plates, the activities of S. cerevisiae Brf1 and Bdp1 are SNAP . The type 1 5S promoters and type 2 promoters surprisingly resistant to deletions. The N-terminal half both use TFIIIC, but on the 5S promoters TFIIIC is re- of ScBrf1 forms an unstable TFIIIB complex but is nev- cruited through the specificity factor TFIIIA. TFIIIA is ertheless capable of directing TFIIIC-independent tran- the founding member of the C H zinc finger family of 2 2 scription from a TATA box if high amounts of ScBdp1 DNA-binding proteins (Miller et al. 1985) and contains are supplied (Kassavetis et al. 1997). The C-terminal half nine C H zinc fingers. In S. cerevisiae, the only essen- 2 2 on its own shows little or no transcription activity, but tial role of TFIIIA is in the transcription of the 5S RNA when the N-terminal half of ScBrf1 is added in trans, genes, because strains engineered to express the 5S peptides encompassing region II mediate high levels of rRNA from a tRNA-type promoter and lacking TFIIIA transcription. Perhaps most surprising, an ScBrf1 protein are viable (Camier et al. 1995). This may explain in part lacking the first 164amino acids including the zinc- the rapid evolution of TFIIIA: TFIIIA sequences from binding domain and the first TFIIB-related repeat retains various organisms are poorly conserved, even among ver- up to 25% of the activity of full-length ScBrf1 for TFIIIC- tebrates. As an example, human and X. laevis TFIIIAs independent transcription from supercoiled templates in share 61% identity over a 264-amino-acid region—of 423 vitro (Kassavetis et al. 1997). Importantly, however, none and 344 amino acids for the human (Arakawa et al. 1995) of these ScBrf1 truncations function in vivo or for and X. laevis (Ginsberg et al. 1984) proteins, respec- TFIIIC-dependent transcription in vitro. Moreover, they tively—whereas the RNA polymerase II transcription do not function for TFIIIC-independent transcription in factor TFIIB is 94% identical in the two species over its vitro from a linear template, suggesting that they are entire length. TFIIIA binds directly to the ICR of type 1 somehow defective in promoter opening. Indeed, with promoters. TFIIIA also binds to 5S RNA to form the 7S the ScBrf1 protein lacking the first 164amino acids, storage ribonucleoprotein particle (Pelham and Brown RNA polymerase III is recruited on a linear template, but 1980). It is present in massive amounts in immature X. the transcription bubble does not form (Kassavetis et al. laevis oocytes, because they accumulate 5S RNA for 1998a). This is probably caused at least in part by the later use during oogenesis and the first rounds of embry- absence of the ScBrf1 zinc ribbon, because point muta- onic cell division, which occur at a rapid pace in the tions within the zinc domain show defects in promoter absence of transcription. This allowed early on the puri- opening as determined by sensitivity to potassium fication of TFIIIA to near homogeneity; indeed, X. laevis permanganate (Hahn and Roberts 2000). Therefore, in TFIIIA was the first eukaryotic transcription factor to be ScBrf1, the zinc ribbon, which is not required for poly- purified (Engelke et al. 1980) and the first for which a merase recruitment, plays a role at a later stage, during corresponding cDNA was isolated (Ginsberg et al. 1984). promoter opening. Upon binding of X. laevis TFIIIA to the 5S gene, the The ScBdp1 TFIIIB subunit also plays a post-RNA TFIIIA zinc fingers are aligned over the length of the ICR with the C-terminal zinc finger in proximity of the 5 polymerase III recruitment role. Thus, ScBdp1 molecules end, and the N-terminal finger in proximity of the 3 end, lacking the conserved region upstream of the SANT of the ICR (for references, see Paule and White 2000). domain (see Fig. 4) can direct somewhat reduced levels Zinc fingers 1–3, which contact the C box, have been of transcription from supercoiled templates but are reported to contribute most of the binding energy of the inactive with linear templates and fail to generate entire protein (Clemens et al. 1992; Liao et al. 1992). permanganate sensitivity around the transcription start Interestingly, however, like TFIIIA fragments containing site (Kassavetis et al. 1998a). Moreover, ScBdp1 is fingers 1–3, fragments containing fingers 4–9 bind, in dispensable for transcription altogether under conditions in which promoter opening is not required (Kassavetis this case to the A box and intermediate element, with et al. 1999). Upon recruitment of RNA polymerase III, affinities approaching that of the full-length protein the SUP4 tRNA gene promoter opens in two seg- (Liao et al. 1992; Kehres et al. 1997). This observation, as ments, one surrounding the transcription start site and well as the binding behavior of full-length proteins with the other located ∼ 7 bp upstream (Kassavetis et al. 1992). zinc fingers mutated either singly or in pairs, suggest With templates containing preformed bubbles extending that simultaneous binding by all nine TFIIIA zinc fingers from −9 to −5, TBP and ScBrf1 alone are sufficient to DNA requires energetically unfavorable distortions, to recruit RNA polymerase III and direct multiple rounds either in the DNA, the protein, or both. Thus, there is of transcription, although the efficiency is only 5% negative cooperativity between certain zinc fingers such to 10% of that observed with the complete TFIIIB com- that loss of binding by a subset of zinc fingers has only a plex. Thus, ScBdp1 plays an essential role in promoter small negative effect on the overall stability of the com- opening. plex (Kehres et al. 1997). Although TFIIIA on its own is GENES & DEVELOPMENT 2603 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez displaced from DNA upon passage of RNA polymerase 1989). These results suggest a factor consisting of two III, the unusual TFIIIA binding properties may contrib- DNA-binding modules separated by a flexible linker that ute to the resilience of the complete 5S transcription can accommodate variously spaced A and B boxes. complex to repeated passage of the RNA polymerase (Bo- genhagen et al. 1982; Setzer and Brown 1985; Wolffe et al. 1986; Darby et al. 1988; Kehres et al. 1997). Yeast TFIIIC Surprisingly little is known about how TFIIIA recruits S. cerevisiae TFIIIC consists of six subunits, Tfc3/138, TFIIIC to the DNA. X. laevis TFIIIA contains a 14- Tfc4/131/PCF1, Tfc1/95, Tfc6/91, Tfc8/60, and Tfc7/ amino-acid domain located C-terminal of the ninth zinc 55, all of which have been cloned and shown to be es- finger, and thus located toward the 5 end of the ICR in sential for cell viability (Willis et al. 1989; Swanson et al. the TFIIIA/5S gene complex, that is dispensable for DNA 1991; Lefebvre et al. 1992; Marck et al. 1993; Arrebola et binding but essential for transcription (Mao and Darby al. 1998; Manaud et al. 1998; Deprez et al. 1999). Five S. 1993). In S. cerevisiae TFIIIA, a hydrophobic segment pombe proteins have recently been identified by BLAST within an 84-amino-acid region located between zinc searches with S. cerevisiae TFIIIC subunits as the query, fingers 8 and 9 is similarly required for cell viability and and four of them confirmed as TFIIIC subunits by im- transcription but not for DNA binding (Rowland and Se- munoaffinity purification of a functional TFIIIC com- gall 1998). These protein domains may play a role in the plex from cells expressing the tagged polypeptides recruitment of TFIIIC. (Huang et al. 2000). These four subunits, referred to as Sfc1, Sfc3, Sfc4, and Sfc6, are orthologs of S. cerevisiae Tfc1, Tfc3, Tfc4, and Tfc6, respectively, as shown in Recruitment factors: TFIIIC Table 2. The fifth one, referred to as Sfc9, shares se- The transcription factor TFIIIC is capable of recognizing quence homology with the S. cerevisiae Tfc8 subunit the TFIIIA–ICR complex on type 1 5S RNA promoters within a short C-terminal segment and may, therefore, and the variously spaced A and B boxes on type 2 tRNA correspond to the S. pombe ortholog of Tfc8. The iden- promoters. The structure of the factor is uniquely tification of these S. pombe subunits is very interesting adapted to perform these tasks. Proteolysis studies indi- because, as detailed below, it clarifies in some cases the cate that S. cerevisiae TFIIIC consists of two domains relationship between S. cerevisiae and human TFIIIC separated by a flexible linker, one of which, designated subunits (Huang et al. 2000). , binds strongly to the B box and the other, designated , binds weakly to the A box (Marzouki et al. 1986). Depending on the distance separating the A and B boxes, Human TFIIIC the factor is visualized by scanning electron microscopy as either two tightly packed or two clearly separated The human TFIIIC fraction contains several activities, globular domains of roughly similar sizes (Schultz et al. some of which are not yet completely defined. These are Table 2. Saccharomyces cerevisiae TFIIIC components andorthologues in Schizosaccharomyces pombe and Homo sapiens S. cerevisiae S. pombe TFIIIC TFIIIC H. sapiens TFIIIC2a Comments Tfc3/ Sfc3 TFIIIC220/TFIIIC Tfc3 and Sfc3 are related, but neither shows sequence similarity to TFIIIC220. Tfc3 cooperates with Tfc6 for binding to DNA. Fragments of TFIIIC220 and TFIIIC110 form a subcomplex capable of binding to the B-box. Tfc4/ /PCF1 Sfc4TFIIIC102/TFIIIC Tfc4protrudes upstream of the start site. TPRs. Tfc4contacts ScBrf1, ScB dp1, and ABC10. Most conserved of the TFIIIC subunits. TFIIIC102 associates with HsBrf1, HsTBP, TFIIIC63. Tfc1/ Sfc1 TFIIIC63/TFIIIC tRNA A-box binding. Tfc1 and Tfc7 associate and can form a distinct complex. TFIIIC63 associates with TFIIIC102, HsBrf1, HsTBP, and HsRPC62. Tfc6 ( ) Sfc6 TFIIIC110/TFIIIC Binds terminator. HMG-I and HMG-Y motifs, WD-40 repeats. Similarity between Tfc6 and TFIIIC110 apparent only through Sfc6. Tfc6 cooperates with Tfc3 for binding to DNA. TFIIIC110 and TFIIIC220 form a subcomplex capable of binding to the B-box. Full-length TFIIIC110 absent in TFIIIC2b. TFIIIC110 possesses HAT activity. Tfc8 ( ) Sfc9 TFIIIC90/TFIIIC Tfc8 bridges  and  domains as well as TFIIIB. Associates with ScTBP. 60 B A Similarity of Tfc8 and Sf9 limited to short C-terminal segment. No similarity between TFIIIC90 and the yeast proteins, but TFIIIC90 binds to TFIIIC220, 110, 63, HsBrf1, HsRPC62, and HsRPC39, and thus may be a functional homolog of the yeast proteins. TFIIIC90 displays HAT activity for histone H3 Lys 14. Tfc7 ( ) none none tRNA A box binding. Tfc7 and Tfc1 associate and can form distinct complex. 2604 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors summarized in Figure 8. The TFIIIC fraction was origi- TFIIIC110/TFIIIC, and TFIIIC90/TFIIIC (see Table nally separated into two fractions, TFIIIC1 and TFIIIC2, 2), whereas TFIIIC2b, which represents 10%–20% of to- that were both required for transcription from the Ad2 tal TFIIIC2 in actively dividing HeLa cells, lacks the VAI gene (Yoshinaga et al. 1987). The TFIIIC1 fraction is TFIIIC110 subunit and appears to contain a 77-kD sub- only partially defined and is discussed further below. unit absent in TFIIIC2a (see Fig. 8; Yoshinaga et al. 1989; The activity in the TFIIIC2 fraction seems to correspond Kovelman and Roeder 1992; Sinn et al. 1995). The nature in part to that of the yeast TFIIIC complex and resides in of the 77-kD subunit is not clear; it is not recognized by a complex of five polypeptides referred to as TFIIIC2 or antibodies generated against the last 595 amino acids of TFIIIC2a (Yoshinaga et al. 1989; Kovelman and Roeder TFIIIC110, suggesting that it either corresponds to an 1992). Human TFIIIC2a has, however, some added ac- unrelated protein or to a TFIIIC110 fragment devoid of tivities compared with S. cerevisiae TFIIIC (see Table 2). epitopes recognized by the antibody. Thus, in cell lines expressing a tagged TFIIIC2a subunit, The TFIIIC220 subunit functionally corresponds to TFIIIC2a can be purified by immunoprecipitation as part the S. cerevisiae Tfc3 subunit because, like Tfc3, it rec- of a larger complex called holo-TFIIIC (see Fig. 8), which ognizes the B box (see below; Table 2). Very strikingly, is described together with the TFIIIC1 fraction further however, it shares no sequence similarity with either S. below (Wang and Roeder 1998; Wang et al. 2000). Holo- cerevisiae Tfc3 or S. pombe Sfc3p (L’Etoile et al. 1994; TFIIIC can bind to chromatin templates, relieves the Lagna et al. 1994; Huang et al. 2000). Similarly, the hu- chromatin-mediated repression of RNA polymerase III man TFIIIC90 protein (Hsieh et al. 1999a) has no obvious transcription from a tRNA gene, and displays histone sequence homolog in S. cerevisiae, but the set of TFIIIC acetyltransferase (HAT) activity (Kundu et al. 1999), and TFIIIB subunits it interacts with suggest that it is the which can be attributed to the intrinsic HAT activity of functional equivalent of Tfc8 (see below). TFIIIC90 has, at least two of the TFIIIC2a subunits (Hsieh et al. 1999a; however, the added property of an intrinsic HAT activity Kundu et al. 1999). for both free and nucleosomal histone H3, and preferen- Besides the active TFIIIC2a complex, the TFIIIC2 frac- tially acetylates histone H3 Lys 14(Hsieh et al. 1999a). tion contains a transcriptionally inactive TFIIIC2 com- The TFIIIC110 subunit corresponds to the yeast Tfc6p plex designated TFIIIC2b (Fig. 8; Kovelman and Roeder protein, although the similarity between the two pro- 1992). TFIIIC2a consists of five subunits referred to as teins is apparent only when compared with the S. pombe TFIIIC220/TFIIIC, TFIIIC102/TFIIIC, TFIIIC63/TFIIIC, ortholog (Huang et al. 2000). Like TFIIIC90, TFIIIC110 Figure 8. Schematic representing the components identified in the human TFIIIC fraction. Holo-TFIIIC was purified by immunoaf- finity from a cell line expressing tagged TFIIIC220 (Wang and Roeder 1998). It is likely, therefore, that it contains TFIIIC2b as well as TFIIIC2a, although this has not been directly demonstrated. See text for references. GENES & DEVELOPMENT 2605 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez has intrinsic HAT activity, and acetylates free and cross-linking (Gabrielsen et al. 1989; Yoshinaga et al. nucleosomal histones H3 and H4as well as nucleosomal 1989; Bartholomew et al. 1990, 1991; Braun et al. 1992a; histone H2B (Kundu et al. 1999). In contrast to the hu- Kovelman and Roeder 1992) and protein–protein associa- man TFIIIC subunits just described, the TFIIIC102 and tion experiments (Shen et al. 1996; Manaud et al. 1998; TFIIIC63 subunits are very clearly related to their yeast Hsieh et al. 1999a,b), TFIIIC models such as those shown counterparts, Tfc4and Tfc1, respectively (Hsieh et al. in Figure 9 can be drawn. In Figure 9, the main sites of 1999b). Thus, human TFIIIC2a is surprisingly divergent cross-linking between yeast subunits and DNA are indi- from S. cerevisiae TFIIIC in all but two of its subunits. cated with dots. Known protein–protein contacts among As detailed further below, these two conserved subunits TFIIIC subunits are indicated by dashes according to are located close to the transcription start site and inter- whether they were demonstrated with human (black) or act directly with TFIIIB subunits. yeast (gray) subunits. On the SUP4 tRNA gene, the S. cerevisiae Tfc3 sub- unit cross-links primarily just upstream of the B box and Assembly of a stable initiation complex on type 1 Tfc6 cross-links at the end of the gene (Bartholomew and 2 promoters et al. 1990, 1991). The two proteins probably cooper- ate to bind to DNA because a mutation in Tfc6 can al- TFIIIC performs at least three functions: It recognizes leviate the binding defect of a Tfc3 mutant (Arrebola et promoter elements, either directly in the case of type 2 al. 1998). Consistent with Tfc3 and Tfc6 corresponding promoters or with the help of TFIIIA in the case of type to human TFIIIC220 and TFIIIC110, respectively, hu- 1 promoters; it recruits TFIIIB; and it contributes to the man TFIIIC220 can be cross-linked to the B box (Yoshi- recruitment of RNA polymerase III. For all of these func- naga et al. 1989; Kovelman and Roeder 1992). Moreover, tions, we have a good idea of which of the TFIIIC sub- TFIIIC220 does not bind DNA on its own, but it is part units are involved. of TFIIIC subassemblies generated by proteinase C cleav- age during poliovirus infection that are still capable of Binding of TFIIIC to type 2 and type 1 promoters binding to DNA (Clark et al. 1991). Some of these sub- A recombinant complex has not yet been reconstituted complexes consist of just the N-terminal 83 kD of either from yeast or human cells, but from both photo- TFIIIC220 associated with the TFIIIC110 subunit or a Figure 9. The position of the various Sc TFIIIC and TFIIIB subunits on an SUP4 tRNA and 5S gene as determined by cross-linking and protein–protein association experiments (see text) are indicated. For the TFIIIC subunits, the names of both the Saccharomyces cerevisiae and the human subunits are indicated. The positions of the start site, end of the gene, and A, B, and C boxes are indicated. A bend in the DNA at the TFIIIB binding site is not illustrated in the figure. The colored dots indicate major cross-linking sites of the subunit of matching color to the DNA. The Tfc8 subunit is shown as an elongated shape extending over most of the TFIIIC complex because it appears to bridge the TFIIIC  and  domains as well as TFIIIB. The black and gray rectangles illustrate protein–protein A B contacts identified with human and S. cerevisiae TFIIIC subunits, respectively. Protein–protein contacts between TFIIIC and TFIIIB subunits are summarized in Figure 10A. 2606 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors fragment thereof, indicating that the DNA-binding do- to sites around and upstream of the start site of tran- main of TFIIIC220 lies within the N-terminal region of scription (Braun et al. 1992a). the protein and that TFIIIC220 and TFIIIC110 are suffi- cient to generate the DNA-binding surface of the TFIIIC2a complex (Shen et al. 1996). Note that an essen- TFIIIC recruitment of TFIIIB tial role for the TFIIIC110 subunit in DNA binding im- plies that the TFIIIC2b complex, which can bind DNA The role of TFIIIC in the recruitment of TFIIIB has been specifically, contains either an unrecognized TFIIIC110 intensively studied in S. cerevisiae. Experiments in fragment or another protein (e.g., the 77-kD protein) pro- which various TFIIIB components were added sequen- viding the TFIIIC110 DNA-binding function. tially to a TFIIIC–DNA complex suggest that the TFIIIC– The yeast Tfc1 and Tfc7 subunits have strong cross- DNA complex interacts initially with the Brf1 compo- links within and near the 3 end of the A box, respec- nent of TFIIIB (Kassavetis et al. 1992), most probably tively (Bartholomew et al. 1990, 1991). Tfc7 interacts through the Tfc4subunit, which protrudes upstream of directly with Tfc1 through its C-terminal half, and the the transcription start site (see Fig. 9). Like the interac- two proteins are not only part of TFIIIC but also form a tion of TFIIIC with its bipartite binding site on DNA, the separate complex in yeast cells (Manaud et al. 1998). interaction of TFIIIC with TFIIIB is highly flexible. Thus, Tyr Tfc8 does not cross-link to DNA. When TFIIIC is treated when TFIIIB is recruited by TFIIIC on SUP4 tRNA with protease, Tfc8 is found in the  domain as deter- gene constructs with TATA boxes inserted at various mined by antibody supershift experiments. In addition, positions upstream of the A box and TFIIIC is subse- however, the temperature sensitivity observed with quently stripped from the DNA by heparin treatment, strains expressing an epitope tagged version of Tfc8 is the upstream and downstream borders of the footprint specifically suppressed by overexpression of Tfc1, which occasioned by the remaining TFIIIB vary according to the resides in the  domain. It is also suppressed by over- position of the TATA box. On the same constructs, the expression of TBP and ScBdp1, and Tfc8 associates with footprint of TFIIIC alone is invariant, but the footprint TBP in vitro (Deprez et al. 1999). Human TFIIIC90 is attributable to TFIIIC (heparin-sensitive) in the presence thought to be the functional homolog of Tfc8 because it of TFIIIB extends to cover the interval between the bor- interacts with TFIIIC220, TFIIIC110, and TFIIIC63, as ders of the variant TFIIIB footprint and that of the invari- well as with the TFIIIB subunit HsBrf1 (Hsieh et al. ant TFIIIC footprint observed with TFIIIC alone. Thus, 1999a). Thus Tfc8/TFIIIC90 appears to bridge the TFIIIC the placement of TFIIIB is codirected by the TATA box and  domains as well as TFIIIB, and is represented, (and thus probably the TBP subunit of TFIIIB) and B A therefore, as extending over the entire gene in Figure 9. TFIIIC. Furthermore, because the interval protected is The Tfc4subunit cross-links to sites around and up- the region to which the Tfc4subunit of TFIIIC cross- stream of the transcription start site (Bartholomew et al. links, these results suggest that Tfc4can extend to ac- 1990, 1991) and directly contacts both the ScBrf1 and commodate various spacings between TFIIIB and TFIIIC ScBdp1 subunits of TFIIIB (Khoo et al. 1994; Chaussivert (Joazeiro et al. 1996). et al. 1995; Rüth et al. 1996; Dumay-Odelot et al. 2002). Tfc4contains 11 copies of the tetratricopeptide repeat Its location on the DNA is consistent with the human (TPR) in four blocks of 5, 4, 1, and 1 repeats (Marck et al. ortholog, TFIIIC102, interacting with TFIIIC63, TBP, 1993; Rameau et al. 1994; Dumay-Odelot et al. 2002). and HsBrf1 (Hsieh et al. 1999b). Tfc1/TFIIIC63 is shown TPRs are found in a large number of proteins including extending upstream of the transcription start site be- subunits of the anaphase-promoting complex and the cause the human subunit, like TFIIIC102, contacts transcription repressor Ssn6. The crystal structures of HsBrf1 and TBP (Hsieh et al. 1999b). several TPR domains indicate that each TPR consists of On the 5S RNA genes, S. cerevisiae TFIIIA cross-links a pair of antiparallel -helices, and that adjacent TPRs strongly to the A box and much more weakly to posi- are packed in regular series of antiparallel -helices (Das tions as far upstream as +20 and as far downstream as et al. 1998; Scheufler et al. 2000). Truncated Tfc4pro- +127, and thus extends over a large portion of the gene teins encompassing the N-terminal, middle, and C-ter- (only the cross-links on the A box are shown in Fig. 9; minal third of the protein including the first (repeats Braun et al. 1992a). TF3C appears shifted downstream as 1–5), second (repeats 6–9), and last (repeats 10 and 11) compared with its position in the tRNA gene, with a blocks of TPRs, respectively, all bind to ScBrf1 in a GST main cross-link at the 3 end of the C box and another pull-down assay (Khoo et al. 1994). Quantitative in vitro one further downstream. As in the tRNA gene, the Tfc6 equilibrium binding assays with various truncated forms subunit cross-links at the end of the gene. There is no of Tfc4also detect several fragments capable of binding indication that the Tfc7 subunit contacts DNA in the 5S to ScBrf1, of which the two with the highest affinities gene, but the Tfc1 subunit cross-links strongly upstream extend from the N terminus of the protein to the end of of the A box. Thus, the 5S A box is clearly not the func- repeat 5, and from repeat 6 to repeat 9 (Moir et al. 2002a). tional equivalent of the tRNA A box. Rather, the func- Interestingly, both fragments have higher affinities for tional equivalent of the tRNA A box in the 5S gene is the ScBrf1 than a larger fragment extending from the N ter- site of Tfc1 cross-linking, which in both genes occurs minus to repeat 9 (Moir et al. 2002a), consistent with the about 30 nt downstream of the transcription start site. observation that in a yeast two-hybrid assay, a similar As in the SUP4 tRNA gene, the Tfc4subunit cross-links long Tfc4fragment shows weaker association than frag- GENES & DEVELOPMENT 2607 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez ments shorter at the C terminus (Chaussivert et al. (Hsieh et al. 1999b). Tfc8 associates with ScBdp1 and 1995). These results suggest that there is autoinhibition ScTBP (Deprez et al. 1999), and the human ortholog for ScBrf1 binding within Tfc4, which is probably re- TFIIIC90 with HsBrf1 (Hsieh et al. 1999a). TFIIIC63 as- lieved by conformational changes during binding. sociates with both HsBrf1 and HsTBP (Hsieh et al. Conformational changes within Tfc4are also sug- 1999b). Thus, there is a network of protein–protein con- gested by an analysis of two dominant Tfc4mutations, tacts between TFIIIC and TFIIIB, all of which may par- PCF-1 and PCF-2, that map within repeats 1–3. These ticipate in the recruitment of TFIIIB during transcription mutations increase transcription both in vivo and in initiation. vitro by increasing the recruitment of TFIIIB, specifically the binding of Tfc4to ScBrf1 (Rameau et al. 1994; Moir TFIIIC contacts RNA polymerase III et al. 1997, 2002b). For PCF1-1, biochemical studies in- dicate that this effect is achieved via a conformational Several of the TFIIIC subunits have been shown to in- change in Tfc4that overcomes autoinhibition in the teract directly with RNA polymerase III subunits, and ScBrf1 binding reaction (Moir et al. 2000, 2002a,b). Thus, these interactions are depicted in Figure 10B with solid the TPRs appear to be involved in conformational arrows for associations demonstrated with human sub- changes that promote association with ScBrf1 and ac- units and hatched arrows for associations demonstrated commodate the variable placement of TFIIIB. The posi- with S. cerevisiae subunits. The Tfc4TFIIIC subunit in- tive effect of the mutations indicates that the recruit- teracts with the RNA polymerase III C53 subunit in a ment of TFIIIB is a limiting step in vivo, and that the two-hybrid assay (Flores et al. 1999), and with the con- conformational change may therefore serve a regulatory served C-terminal domain of the universal RNA poly- role. merase subunit ABC10 in both a yeast two-hybrid assay Other associations have been observed between iso- and in vitro. This latter interaction is likely to be func- lated TFIIIC and TFIIIB subunits. These are symbolized tionally significant because a thermosensitive mutation in Figure 10A with arrows, according to whether the as- within the C-terminal domain of ABC10 that weakens sociation was demonstrated with human (solid) or yeast the interaction can be rescued in an allele-specific man- (hatched) TFIIIC subunits. As just described, Tfc4asso- ner by overexpression of a variant form of Tfc4that ciates with ScBrf1, and it also associates with ScBdp1 strengthens the interaction (Dumay et al. 1999). Among (Khoo et al. 1994; Chaussivert et al. 1995; Rüth et al. the human TFIIIC subunits, the TFIIIC90 protein inter- 1996; Dumay-Odelot et al. 2002). The human ortholog acts with HsRPC62 and HsRPC39, which are both part TFIIIC102 associates with both HsBrf1 and HsTBP of the subcomplex of RNA polymerase III subunits re- Figure 10. Protein–protein contacts between TFIIIC and TFIIIB subunits, and TFIIIC and RNA polymer- ase III subunits. Contacts as determined either by in vitro or yeast two-hybrid assays are depicted. Ge- netic interactions have not been included. Solid ar- rows depict interactions shown with human pro- teins, stippled arrows depict interactions shown with Saccharomyces cerevisiae proteins. See text for references. (A) Protein–protein contacts between TFIIIC and TFIIIB subunits. (B) Protein–protein con- tacts between TFIIIC and RNA polymerase III sub- units. 2608 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors quired specifically for transcription initiation, and and SNAP45 can each associate independently with TFIIIC63 associates with HsRPC62 in vitro (Hsieh et al. SNAP190, and SNAP43 can associate independently with 1999a). These TFIIIC–RNA polymerase III interactions SNAP50. SNAP43 can also associate with SNAP190, but emphasize that TFIIIC not only recruits TFIIIB but may in coimmunoprecipitations of in vitro translated pro- also participate in the recruitment of RNA polymerase teins, this association is not detectable unless SNAP19 is III. Thus, although TFIIIB is sufficient to recruit RNA present. This suggests that SNAP43 has weak contacts polymerase III, in the cell both TFIIIB and TFIIIC are with both SNAP190 and SNAP19, only the sum of which likely to work in concert to recruit the RNA polymerase. is measurable by the stringent coimmunoprecipitation assay (Henry et al. 1998b). However, partial complexes containing SNAP190 or a truncated SNAP190, SNAP43, Recruitment factors: SNAP and SNAP50 can be assembled both in insect cells in- fected with recombinant baculoviruses (Mittal et al. Type 3 promoters do not contain binding sites for TFIIIA 1999) and from subunits overexpressed in E. coli (Ma and and/or TFIIIC2, and, indeed, they do not require TFIIIC2 Hernandez 2000, 2002), indicating that in these situa- for assembly of a transcription initiation complex. Thus, tions SNAP19 is dispensable for complex assembly. depletion of a transcription extract with antibodies di- The domains involved in protein–protein contacts are rected against the DNA-binding subunit of TFIIIC2, summarized in Figure 11B. Within SNAP190, amino ac- TFIIIC220, affects transcription from the 5S, tRNA, and ids 84–133 are sufficient for association with SNAP19 VAI genes but not from the 7SK and U6 alone, and with SNAP43 together with SNAP19. This snRNA genes (Lagna et al. 1994). Instead, the gene-ex- SNAP190 region, and the N-terminal part of SNAP19, ternal PSE of type 3 promoters recruits the multisubunit are likely to form -helices and may be involved in a complex SNAP . SNAP is a complex containing five c c coiled-coil type of interaction with each other. In different subunits, SNAP190 (Wong et al. 1998), SNAP43, amino acids 164–268 are sufficient for associa- SNAP50/PTF (Bai et al. 1996; Henry et al. 1996), tion with SNAP190 together with SNAP19, and amino SNAP45/PTF (Sadowski et al. 1996; Yoon and Roeder acids 1–163 are sufficient for association with SNAP50. 1996), SNAP43/PTF (Henry et al. 1995; Yoon and Thus, these two association domains in SNAP43 are Roeder 1996), and SNAP19 (Henry et al. 1998b). In the completely separable. The SNAP190 region required for human system, SNAP is involved in transcription of interaction with SNAP45 lies between amino acids 1281 snRNA genes by both RNA polymerases II and III, as depletion of endogenous SNAP from transcription ex- and 1393. The SNAP190 regions required for association tracts debilitates U1 and U6 transcription and transcrip- with SNAP19/SNAP43 and with SNAP45 thus lie at op- tion can be restored in both cases by addition of highly posite ends of the linear molecule (Ma and Hernandez purified recombinant SNAP (Henry et al. 1998b). 2000). The subunit–subunit contacts within SNAP have SNAP binds specifically to the PSE. This binding is c c been determined by reconstitution of partial complexes mediated in part by an unusual Myb domain extending and coimmunoprecipitations of various subsets of in from amino acids 263 to 503 within SNAP190, with four vitro translated full-length or truncated SNAP subunits and one-half Myb repeats designated the Rh (R half), Ra, (Henry et al. 1996, 1998a,b; Wong et al. 1998; Mittal et Rb, Rc, and Rd repeats (see Fig. 11B; Wong et al. 1998). al. 1999; Ma and Hernandez 2000, 2002). Figure 11A Indeed, a SNAP190 segment consisting of just the Myb shows the general architecture of the complex. SNAP19 domain can bind to the PSE (Wong et al. 1998), and in the Figure 11. Subunit–subunit interactions within SNAP .(A) For simplicity, the complex is shown as containing one copy of each subunit, but the stoichiometry of the SNAP subunits has not been determined. (B) The domains of the various subunits sufficient for association with other subunits in a coimmunoprecipitation assay. The TBP recruitment region 1 (TRR1), the Myb domain with the half repeat (Rh) followed by four repeats (Ra, Rb, Rc, and Rd), and the Oct-1 interacting region (OIR) are shown. GENES & DEVELOPMENT 2609 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez context of the full complex, the last two Myb repeats (Rc and Rd), but not the first two and one-half (Rh, Ra, and Rb), are required for binding to the PSE (Mittal et al. 1999). However, even though the Myb domain is re- quired, it is not sufficient. Thus, although a SNAP190 segment consisting of just the Myb domain binds DNA, larger SNAP190 segments do not. The smallest subas- sembly of SNAP subunits characterized that binds spe- cifically to DNA consists of SNAP190 amino acids 84– 505, SNAP43 amino acids 1–268, and SNAP50 (complex #8; Ma and Hernandez 2000, 2002). Consistent with the requirement for parts of SNAP190 and SNAP50 for DNA binding, UV cross-linking experiments suggest that both SNAP190 (Yoon et al. 1995) and SNAP50 (Henry et al. 1996) are in close contact with DNA. Figure 12. Human U6 transcription initiation complex. TBP (yellow) and SNAP (orange) bind cooperatively to the DNA, presumably through a direct protein–protein contact that in- Assembly of a stable transcription complex on the type volves a 50-amino-acid segment within the N-terminal region 3 U6 snRNA promoter of SNAP190. TBP also binds cooperatively with Brf2. SNAP and the Oct-1 POU domain (blue) bind cooperatively to DNA, As noted above, the type 3 promoters such as the U6 through a direct protein–protein contact involving a glutamic promoter contain, in addition to core sequences, a DSE acid at position 7 within the POU domain (blue triangle) and a that serves to enhance transcription from the core pro- lysine at position 900 within SNAP190 (red triangle). This di- moter (see Fig. 1). The DSE can contain a number of rect protein–protein interaction is mediated by a positioned protein-binding sites, but two of them are almost invari- nucleosome (green) that brings into close proximity the octamer sequence and the PSE. Adapted from Zhao et al. (2001). See text ably an SPH (also called NONOCT) element, which re- for references. cruits the transcription factor STAF (also called SPH- binding factor or SBF), and an octamer sequence, which recruits the transcription factor Oct-1 (for reviews, see Hernandez 1992, 2001). is efficiently recruited to the DNA through at least two STAF is a zinc finger protein, and Oct-1 is a founding cooperative binding interactions. SNAP binds coopera- member of the POU-domain protein family. Both pro- tively with TBP, which like SNAP binds poorly to DNA teins contain two types of activation domains, one type on its own, and with the Oct-1 POU domain (Murphy et that specifically activates mRNA-type RNA polymerase al. 1992; Mittal et al. 1996; Mittal and Hernandez 1997). II promoters and another that specifically activates both In cooperative binding of SNAP and TBP, both factors type 3 RNA polymerase III promoters and the very simi- recruit each other to the DNA. Mini-SNAP is also able lar RNA polymerase II snRNA promoters (Tanaka et al. to bind cooperatively with TBP, although in this case, 1992; Das et al. 1995; Schuster et al. 1998). The presence cooperative binding results mainly in the recruitment of of the latter type of activation domains in STAF and TBP, because mini-SNAP binds efficiently to DNA on Oct-1 supports the idea that these proteins activate type its own (Mittal et al. 1999). Within the context of mini- 3 promoters (as well as RNA polymerase II snRNA pro- SNAP , a 50-amino-acid segment within the N-terminal moters) in the cell. Indeed, both proteins have been lo- region of SNAP190, called TRR1 (TBP recruitment re- calized to snRNA promoter sequences in vivo by chro- gion 1; see Fig. 11) is required for cooperative binding matin immunoprecipitation experiments (Zhao et al. with TBP. Interestingly, mini-SNAP complexes lacking 2001; Mach et al. 2002). Thus, as for the core promoter these 50 amino acids are active for transcription, sug- sequences of type 3 promoters, the key factors recruited gesting that during assembly of the U6 transcription ini- by the DSE are well characterized and are available in tiation complex, there are redundant mechanisms to re- recombinant form. cruit TBP to the DNA. Indeed, TBP is efficiently re- The identification of the factors binding directly to cruited to the DNA by cooperative binding with HsBrf2 type 3 promoter elements has allowed the characteriza- to form a complex containing mini-SNAP , TBP, and tion of their assembly onto a human U6 promoter. Our HsBrf2 (Ma and Hernandez 2002), and TBP is therefore present understanding of this process is summarized in shown contacting both SNAP and HsBrf2 in Figure 12. Figure 12. SNAP binds poorly to DNA on its own. In- It remains possible that in the absence of the DSE, co- terestingly, however, a partial complex lacking the C- operative binding of TBP and SNAP is essential to re- terminal two-thirds of the largest subunit of SNAP as cruit the full SNAP to the DNA, which, unlike mini- well as SNAP45, referred to as mini-SNAP , binds more SNAP , binds poorly to the PSE. In the presence of the efficiently to the DNA (Mittal et al. 1999). This implies DSE, however, the full SNAP can be recruited to the that the C-terminal two-thirds of SNAP190 and/or DNA by the Oct-1 POU domain. SNAP45, which associates with the C-terminal portion The Oct-1 POU domain is a bipartite DNA-binding of SNAP190, somehow down-regulate binding of the domain consisting of two helix–turn–helix-containing complex to DNA. On the U6 promoter, however, SNAP DNA-binding modules: an N-terminal POU-specific 2610 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors (POU ) domain and a C-terminal POU-homeo domain The recruitment of SNAP by the Oct-1 POU domain S c (POU ), joined by a flexible linker (Herr and Cleary contributes to transcription activation in vitro. There- 1995). The POU domains of Oct-1 and the pituitary tran- fore, Oct-1 is likely to activate snRNA gene transcrip- scription factor Pit-1 are ∼ 50% identical, but only the tion in vivo not only through its transcription activation Oct-1 POU domain is capable of recruiting SNAP to the domains but also through its DNA-binding domain. This PSE. This difference in activity is due to only one of the activation can be compared to activation in prokaryotes many amino acid differences between the two proteins, a by the  cI repressor, which activates transcription from the  P glutamic acid at position 7 in the Oct-1 POU domain promoter by favoring open complex formation S RM changed to an arginine at the corresponding position in through direct protein–protein contacts between its the Pit-1 POU domain. Thus, an E7R mutation in Oct-1 DNA-binding domain and RNA polymerase (for refer- does not affect Oct-1 binding to the octamer sequence ences, see Ford et al. 1998). Strikingly, the DNA-binding but inhibits cooperative binding with SNAP , and an domain of the  cI repressor is structurally similar to the R7E mutation in Pit-1 imparts to this protein the ability Oct-1 POU domain, and the RNA polymerase subunit to bind cooperatively with SNAP (Mittal et al. 1996). contacted by the cI DNA-binding domain is the sub- Oct-1 associates with SNAP190, the largest subunit of unit, which is, like SNAP , a core promoter binding fac- SNAP , both in a yeast one-hybrid assay and in an elec- tor. This illustrates how E. coli and human cells can use trophoretic mobility shift assay (Ford et al. 1998; Wong similar protein–protein contacts to activate transcrip- et al. 1998). This suggested that cooperative binding of tion. the Oct-1 POU domain and SNAP may result from a The Oct-1–SNAP interaction was originally charac- c c protein–protein contact between the POU domain and terized with U6 promoter probes in which the octamer SNAP190. Indeed, a small domain of SNAP190 extend- sequence had been placed close to the PSE. The exact ing from amino acids 869 to 912 and labeled OIR (Oct- distance separating the octamer and the PSE was not 1-interacting region) in Figure 11 above is sufficient for important, but not surprisingly, cooperative binding was association with the Oct-1 POU domain in an electro- not observed on the natural promoter, where the two phoretic mobility shift assay. Strikingly, the OIR of sites are separated by ∼ 150 bp. This suggested that in the SNAP190 displays sequence similarity with the Oct-1 natural U6 promoter the two sites may somehow be POU-domain-interacting region present in OBF-1/ brought into close proximity for cooperative binding. In- OCA-B (Ford et al. 1998). OBF-1 is a B-cell-specific coac- deed, as shown in Figure 12, the human U6 promoter tivator of Oct-1, which recognizes Oct-1 bound to an harbors a positioned nucleosome between the octamer octamer sequence and provides a strong activation do- sequence and the PSE, both in vivo as determined by main of mRNA gene transcription. As with SNAP190, micrococcal nuclease sensitivity of the chromatin and in the association of OBF-1 with the Oct-1 POU domain is vitro upon assembly of a U6 template into nucleosomes sensitive to the E7R mutation in Oct-1 (Babb et al. 1997), (Stunkel et al. 1997; Zhao et al. 2001). The positioned and, indeed, the Oct-1 POU E7 is involved in a hydrogen nucleosome promotes activation by the Oct-1 POU do- bond with a lysine in OCA-B as shown in the crystal main from its natural binding site as well as cooperative structure of an OCA-B peptide associated with an Oct-1 binding of Oct-1 and SNAP . This cooperative binding is POU/octamer sequence complex (Chasman et al. 1999). affected by the Oct-1 E7R and the SNAP190 K900E mu- Thus, two proteins with different tissue distribution, tations, but is restored to significant levels when the two function, and primary structure nevertheless share a mutant factors are combined (Zhao et al. 2001). Thus, common Oct-1 binding domain, and probably contact the positioned nucleosome mediates a direct protein– overlapping surfaces in the Oct-1 POU domain (Ford et protein contact between the Oct-1 POU domain and al. 1998). SNAP , and this contact is the same as the one charac- Within the SNAP190 OIR, mutation of Lys 900 to a terized on probes containing closely spaced octamer and glutamic acid (K900E mutation) inhibits cooperative PSE. This provides a striking example of a nucleosome, binding of SNAP with the Oct-1 POU domain. Very the structural unit of chromatin, activating transcription significantly, however, if SNAP carrying the K900E mu- by serving an architectural role and thus allowing coop- tation within SNAP190 is combined with the Oct-1 erative interactions of factors binding to distant sites. POU domain carrying the E7R mutation, significant co- The assembly of the U6 transcription complex illus- operative binding is restored (Ford et al. 1998). The iso- trates how a network of cooperative interactions ulti- lation of these paired altered specificity mutants indi- mately increases both the affinity and the DNA-binding cates that cooperative binding requires a direct protein– specificity of factors such as SNAP and TBP which, on protein contact, and suggests that this contact involves their own, display slow on and off rates and relatively K900 in SNAP190 and E7 in the Oct-1 POU domain. low DNA-binding specificity. Both SNAP and TBP con- K900 (Fig. 12, red triangle) and E7 (Fig. 12, blue triangle) tain built-in dampers of DNA binding. In the case of are, therefore, shown contacting each other in Figure 12. SNAP , the damper is deactivated by cooperative inter- In SNAP , then, the same region of the complex that is actions with Oct-1, which dramatically enhance the required for down-regulation of DNA binding is involved SNAP on rate (Mittal et al. 1996). In the case of TBP, the in a protein–protein interaction that mediates coopera- damper is deactivated by cooperative interactions with tive binding such that, in effect, cooperative binding in- SNAP (Mittal and Hernandez 1997). Because coopera- activates a SNAP built-in damper of DNA binding. tive interactions between Oct-1 and SNAP , and SNAP c c c GENES & DEVELOPMENT 2611 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Schramm and Hernandez and TBP, only take place when the corresponding DNA- 50, 45, and 40 kD (Wang and Roeder 1998), whose iden- binding sites are all present, such interactions limit, in tities have not yet been reported. This relatively stable effect, the binding of these factors to promoter se- TFIIIC1/TFIIIC2 complex is referred to as human TFIIIC quences. (see Fig. 8). A TFIIIC1 activity purified by conventional chromatography, which may correspond to the four poly- peptides, binds cooperatively with both TFIIIC2 and a Stepwise assembly versus holoenzyme TFIIIC2/TFIIIA complex, and the footprint displayed by TFIIIC2 and the TFIIIC1 activity on a tRNA gene ex- Our understanding of how RNA polymerase III is re- tends over the A and B boxes (Oettel et al. 1997). cruited to specific promoters stems from in vitro studies In addition to the TFIIIC1 factor, the TFIIIC1 fraction in which transcription initiation complexes are as- contains an uncharacterized factor that specifically en- sembled by stepwise addition of various components. hances human U6 transcription and has been called Such studies have brought invaluable information about TFIIICU (Oettel et al. 1998) as well as an activity that the network of protein–DNA and protein–protein inter- footprints, on its own, over the terminator region of the actions that culminates in the specific recruitment of VAI gene and a tRNA gene (see Fig. 8; Wang and Roeder RNA polymerase III. In vivo, however, it is possible that 1996; Oettel et al. 1997, 1998). The latter activity is also many of the factors that mediate promoter recognition present in holo-TFIIIC but can be dissociated from the by RNA polymerase III are recruited together with the TFIIIC factor with 300 mM KCl (Wang et al. 2000). It has polymerase as part of a holoenzyme. Indeed, anti-epitope been purified by a combination of conventional and tag immunoprecipitates from a human cell line stably DNA affinity chromatography to near homogeneity, and synthesizing a tagged subunit of RNA polymerase III corresponds to four groups of NF1 isoforms (Wang et al. can direct transcription from type 2 promoters on their 2000). NF1 was initially identified as a cellular factor own and from type 1 promoters when combined with required for efficient initiation of Ad2 replication (Na- TFIIIA (Wang et al. 1997). Consistent with this observa- gata et al. 1982), and was later found to be involved in the tion, these immunoprecipitates contain the TFIIIB sub- expression of many cellular and viral genes. The purified units TBP and HsBrf1 as well as the TFIIIC2 subunits activity gives rise to a footprint over the two VAI termi- TFIIIC220 and TFIIIC110 as determined by immunoblot. nator regions that is identical to that observed with holo- Tandem immunoprecipitations as well as gel filtration TFIIIC, consistent with the idea that holo-TFIIIC con- analyses indicate that about 10% of the RNA polymer- tains, at a minimum, TFIIIC1, TFIIIC2, and NF1 poly- ase molecules in the immunoprecipitate is associated peptides (Fig. 8). The role of NF1 in RNA polymerase III with TFIIIB and/or TFIIIC components. However, these transcription is further addressed below. associations are reduced by an increase in the KCl con- centration from 100 mM to 150 mM, and are lost at 300 Termination and recycling: does the end help mM KCl. Nevertheless, these results raise the possibility the beginning? that in vivo, RNA polymerase III is recruited together with some of its accessory factors, in particular TFIIIB RNA polymerase III is unique among the eukaryotic and TFIIIC2 (Wang et al. 1997). RNA polymerases in recognizing a simple run of T resi- dues as a termination signal. In a linear template, muta- tion or deletion of the termination signal results in the Human TFIIIC1 production of run-off transcripts. However, aberrant ter- mination is not the only consequence of debilitating the The human TFIIIC fraction contains the B-box-binding terminator. Mutation of the run of T residues in the VAI TFIIIIC2 complex described above as well as the TFIIIC1 gene diminished the efficiency of single- and multiple- activity, which displays no strong DNA-binding activity round transcription in a HeLa cell extract (Wang and on its own but extends the TFIIIC2 footprint both up- Roeder 1996; Wang et al. 2000). Similarly, in an S. cer- stream on the A box and downstream to the end of the evisiae system in which RNA polymerase III was limit- gene (Yoshinaga et al. 1987). TFIIIC1 is apparently re- ing, deletion of the terminator affected multiple-round quired not only for transcription from type 1 and 2 pro- transcription, although in this case single-round tran- moters, but also for transcription from the type 3 gene- scription was not affected (Dieci and Sentenac 1996). external promoters (Yoon et al. 1995). As summarized in These results suggest that the run of T residues can con- Figure 8, this fraction contains a number of factors that tribute to the efficiency of initiation and reinitiation, have been implicated in RNA polymerase III transcrip- and therefore that it may play a role in RNA polymerase tion, many of which are still poorly characterized. Most recycling. In higher eukaryotes, a number of factors have of the TFIIIC1 activities can be purified as part of a holo- been implicated in efficient termination and RNA poly- TFIIIC complex by immunopurification of TFIIIC2 from merase III recycling, including the La protein, NF1 poly- a cell line expressing tagged TFIIIC220 (Wang and Roeder peptides, DNA topoisomerase I, and PC4. 1998). One of them, called TFIIIC1 (as opposed to the TFIIIC1 fraction), remains associated with TFIIIC2 after La protein further purification of the immunoprecipitated com- plex on a sucrose gradient and consists of at least four The first factor implicated in transcription termination polypeptides with apparent molecular masses of 70, and recycling was the La antigen (Gottlieb and Steitz 2612 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase III and its transcription factors 1989a,b). La binds to the poly-U tail at the end of RNA tive factors not present in the purified system (Wang et polymerase III transcripts (Stefano 1984), and could be al. 2000). Thus, NF1 can increase VAI transcription effi- demonstrated to stimulate transcription in a system in ciency in a crude extract, perhaps by facilitating tran- which transcription complexes were (1) preassembled on scription termination. immobilized templates; (2) allowed to undergo a single Depletion of NF1 reportedly also reduced transcription round of transcription in the presence of heparin and from the 5S RNA, a tRNA, and the human U6 snRNA genes, suggesting that NF1 polypeptides are generally re- sarcosyl, which resulted in stripping of RNA polymerase III; (3) washed; and (4) incubated with either fresh RNA quired for efficient RNA polymerase III transcription in polymerase alone or RNA polymerase together with La. crude extracts (Wang et al. 2000). It is not clear, however, The effect could be attributed to improved transcript re- how NF1 is recruited to other RNA polymerase III ter- lease and transcription reinitiation (Maraia 1996), and minators with no obvious NF1-binding sites close to the was abolished by phosphorylation of Ser 366 in La (Fan et run of T residues. Perhaps in these cases, protein–protein al. 1997). On the other hand, however, depletion of La interactions with TFIIIC2 (or, for genes such as the hu- from crude X. laevis extracts did not affect RNA poly- man U6 snRNA gene that do not recruit TFIIIC2, some merase III transcription (Lin-Marq and Clarkson 1998). other transcription factor) mediate NF1 recruitment. Thus, although La can clearly affect RNA polymerase III transcription in certain in vitro systems, it is not clear that the protein plays such a role in the cell. DNA topoisomerase I andPC4 Like NF1, both DNA topoisomerase I and PC4can sup- NF1 polypeptides press read-through transcripts in the dC-tailed template assay (Wang and Roeder 1998; Wang et al. 2000). How- Other factors implicated in transcription termination ever, these factors seem to differ from NF1 in that even and efficiency are the NF1 polypeptides in the TFIIIC1 though they enhance TFIIIC interactions in the down- fraction, which footprint over the VAI terminator region stream region of a tRNA gene and the VAI gene, they do (Wang et al. 2000). NF1 polypeptides form a family of not generate a footprint on their own, and the footprint proteins with multiple functions encoded by a large observed in the presence of TFIIIC does not appear to number of alternatively spliced RNAs derived from four extend over the termination region (Wang and Roeder different genes. Individual NF1 proteins can dimerize 1998; Wang et al. 2000). DNA topoisomerase I and PC4 with themselves and with other NF1 variants, but all are present in holo-TFIIIC, but unlike NF1, they are pres- variants have the same DNA-binding specificity and rec- ent only in trace amounts. Thus, DNA topoisomerase I ognize the consensus sequence 5-YTGGCANNNTGC and PC4seem less likely than NF1 to participate in RNA CAR-3. Sequences that closely match this consensus polymerase III transcription in vivo. are found in the two VAI terminators, immediately downstream of the run of T residues, and mutations in these NF1-binding sites resulted in the appearance of Conclusion read-through transcripts, although the effect was not as severe as with mutation of the run of T residues. Fur- The study of RNA polymerase III promoters has provided thermore, addition of NF1 together with the TFIIIC fac- perhaps the best illustration of how promoters with dif- tor improved the ratio of correctly terminated transcripts ferent structures rely differently on DNA–protein and over read-through transcripts derived from a dC-tailed protein–protein contacts to recruit, ultimately, the same template, in which RNA polymerase III initiates tran- RNA polymerase. Thus, TFIIIB can be recruited to a scription at the dC tail in the absence of transcription TATA box through direct binding to DNA, or to many initiation factors (Wang et al. 2000). Together, these re- DNA sequences through binding to TFIIIC. TFIIIC itself sults suggest an involvement of NF1 polypeptides in can bind directly to the B box of type 2 promoters or can transcription termination, although the interpretation of use the accessory factor TFIIIA to bind to type 1 promot- the dC-tailed template experiment is complicated by the ers. And in type 3 promoters, TFIIIB is recruited through fact that initiation at tailed templates tends to result in a combination of DNA contacts with the TATA box and protein contacts with SNAP the production of RNA–DNA hybrids, which are known . These promoters also il- to inhibit transcription termination by RNA polymerase lustrate how a transcription machinery has diversified III (Campbell and Setzer 1992). and expanded from S. cerevisiae with its relatively In addition to a qualitative effect on transcription ter- simple genome to higher eukaryotes with their much mination, NF1 polypeptides can affect transcription ef- more complex genomes to include new factors, such as ficiency. Thus, depletion of NF1 polypeptides from an SNAP , or multiple variations of a factor unique in yeast, extract severely reduced VAI transcription, and efficient such as HsBrf1 and HsBrf2. transcription could be restored by addition of either pu- RNA polymerase III transcription is tightly regulated; rified NF1 activity or recombinant CTF1, the largest it is controlled by growth conditions, during the cell NF1 variant derived from the NF1-C gene (Santoro et al. cycle, and by a number of viruses (for review, see Brown 1988). However, in a highly purified transcription sys- et al. 2000). These controls involve RNA polymerase III tem, the stimulatory effect of NF1 was reportedly not transcription factors, in particular TFIIIC2 and TFIIIB, as observed, suggesting that NF1 serves to counteract nega- well as, perhaps, RNA polymerase III itself. 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Delineation of two functional regions of transcription associated with Maf1, a negative regulator of RNA poly- factor TFIIB. Proc. Natl. Acad. Sci. 90: 5628–5632. merase III transcription (Pluta et al. 2001). So far, recon- Bartholomew, B., Kassavetis, G.A., and Geiduschek, E.P. 1990. stitution of RNA polymerase III transcription with com- The subunit structure of Saccharomyces cerevisiae tran- pletely defined (recombinant) transcription factors has scription factor IIIC probed with a novel photocrosslinking reagent. EMBO J. 9: 2197–2205. been achieved only in the S. cerevisiae system with a ———. 1991. Two components of Saccharomyces cerevisiae promoter that can recruit TFIIIB directly through a transcription factor IIIB (TFIIIB) are stereospecifically lo- TATA box. An urgent task, which will allow a detailed cated upstream of a tRNA gene and interact with the second- understanding of the mechanisms by which RNA poly- largest subunit of TFIIIC. Mol. Cell. 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Willis, I.M. 2002. A universal nomenclature for subunits of the RNA polymerase III transcription initiation factor TFIIIB. Genes & Dev. 16: 1337–1338. Willis, I., Schmidt, P., and Soll, D. 1989. A selection for mutants 2620 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Laura Schramm and Nouria Hernandez Genes Dev. 2002, 16: Access the most recent version at doi:10.1101/gad.1018902 This article cites 224 articles, 128 of which can be accessed free at: References http://genesdev.cshlp.org/content/16/20/2593.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Cold Spring Harbor Laboratory Press

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