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A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II

A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 32, Issue of August 6, pp. 22127–22130, 1999 Communication © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. posed of the kinase/cyclin pair CDK7/cyclin H and the A Role for the TFIIH XPB DNA RING-H2 finger protein MAT1. TFIIH subunits are found in a Helicase in Promoter Escape variety of additional subassemblies, including a six-subunit complex (IIH6) containing XPB, XPD, p62, p52, p44, and p34, a by RNA Polymerase II* five-subunit “core” complex (IIH5) containing XPB, p62, p52, p44, and p34, and a four-subunit XPD/CAK complex (2– 6). (Received for publication, May 26, 1999) TFIIH was initially identified by its requirement in tran- Rodney J. Moreland‡, Franck Tirode§, scription initiation by RNA polymerase II (7). Initiation is an ¶ ¶i Qin Yan‡ , Joan Weliky Conaway‡ **, ATP-dependent process that requires at minimum the five Jean-Marc Egly§, and Ronald C. Conaway‡‡‡ general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and From the ‡Program in Molecular and Cell Biology, TFIIH (8, 9). Biochemical studies have shown that initiation in Oklahoma Medical Research Foundation, Oklahoma this minimal transcription system proceeds through multiple City, Oklahoma 73104, the §Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ stages beginning with assembly of polymerase and all five ULP, B. P. 163, Illkirch, C.U. de Strasbourg, France, general initiation factors into a closed preinitiation complex at the Department of Biochemistry and Molecular the promoter (8, 9) and culminating in ATP-dependent forma- Biology, University of Oklahoma Health Sciences tion of the open complex and synthesis of the first phosphodi- Center, Oklahoma City, Oklahoma 73190, and the ester bond of nascent transcripts (10 –13). Evidence supporting Howard Hughes Medical Institute, Oklahoma Medical a role for TFIIH DNA helicase activity in ATP-dependent for- Research Foundation, Oklahoma City, Oklahoma 73104 mation of the open complex was initially suggested by studies TFIIH is an RNA polymerase II transcription factor indicating that both TFIIH and ATP are dispensible for initi- that performs ATP-dependent functions in both tran- ation by RNA polymerase II from artificial promoters contain- scription initiation, where it catalyzes formation of the ing premelted transcriptional start sites and from promoters on open complex, and in promoter escape, where it sup- negatively supercoiled DNA templates (14 –19). presses arrest of the early elongation complex at pro- In addition to its requirement in transcription initiation, moter-proximal sites. TFIIH possesses three known ATP- TFIIH is also required for efficient promoter escape by RNA dependent activities: a 3*3 5* DNA helicase catalyzed by polymerase II (18, 20 –22). Mechanistic studies have shown its XPB subunit, a 5* 3 3* DNA helicase catalyzed by its that a fraction of early RNA polymerase II elongation interme- XPD subunit, and a carboxyl-terminal domain (CTD) ki- nase activity catalyzed by its CDK7 subunit. In this re- diates are prone to arrest at promoter-proximal sites in the port, we exploit TFIIH mutants to investigate the con- absence of TFIIH or an ATP cofactor (18, 21–23). Circumstan- tributions of TFIIH DNA helicase and CTD kinase tial evidence that TFIIH DNA helicase activity is responsible activities to efficient promoter escape by RNA polymer- for suppressing arrest of early elongation intermediates has ase II in a minimal transcription system reconstituted come from the observation that promoter escape is blocked by with purified polymerase and general initiation factors. the TFIIH DNA helicase inhibitor ATPgS, but not by the TFIIH Our findings argue that the TFIIH XPB DNA helicase is CTD kinase inhibitor H-8 (18). primarily responsible for preventing premature arrest Although evidence from previous studies suggested that of early elongation intermediates during exit of polym- TFIIH DNA helicase activity is required for ATP-dependent erase from the promoter. formation of the open complex and ATP-dependent promoter escape, a direct test of this hypothesis was not possible until sufficient quantities of purified TFIIH mutants lacking func- TFIIH is a nine-subunit complex that possesses multiple tional XPB or XPD DNA helicase were available. Recently, catalytic activities, including DNA-dependent ATPase, DNA some of us (F. Tirode and J.-M. Egly) reported the development helicase, and a protein kinase that is capable of phosphoryl- of methods for reconstitution of TFIIH and TFIIH subassem- ating the carboxyl-terminal domain (CTD) of the largest sub- blies from wild type and mutant subunits (2, 4). By investigat- unit of RNA polymerase II (1). The two largest TFIIH subunits ing the activities of TFIIH mutants, we observed that maximal are ATP-dependent DNA helicases encoded by the Xeroderma TFIIH transcriptional activity requires all nine subunits, al- pigmentosum complementation group B (XPB) and D (XPD) though the TFIIH subassembly IIH6 lacking CAK is active in genes. The TFIIH-associated CTD kinase is a three-subunit ATP-dependent formation of the open complex and supports a subassembly, CDK-activating kinase (CAK), which is com- reduced level of runoff transcription (4). In addition, by com- paring the activities of IIH6 and two IIH6 mutants, IIH6/XPB- * This work was supported in part by National Institutes of Health K346R and IIH6/XPD-K48R, which contain point mutations in Grant GM41628 (to R. C. C.), a Human Frontier Grant (to J. M. E.), and the XPB and XPD ATP binding sites and lack DNA helicase by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust. The costs of publi- activity (24, 25), we obtained evidence supporting the model cation of this article were defrayed in part by the payment of page that the XPB DNA helicase is essential for formation of the charges. This article must therefore be hereby marked “advertisement” open complex and runoff transcription and that the XPD DNA in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. helicase, though not essential, stimulates these reactions (2). ** Associate Investigator of the Howard Hughes Medical Institute. ‡‡ To whom correspondence should be addressed. Tel.: 405-271-1950; In this report, we exploit recombinant TFIIH mutants lack- Fax: 405-271-1580. ing functional XPB DNA helicase, XPD DNA helicase, or CAK The abbreviations used are: CTD, carboxyl-terminal domain; 39-O- to investigate the contribution of TFIIH DNA helicase and CTD MeGTP, 39-O-methylguanosine 59-triphosphate; ATPgS, adenosine 59- kinase activities to efficient promoter escape. Our findings O-(thio)triphosphate; AdML, adenovirus 2 major late; CAK, CDK-acti- vating kinase. argue that the XPB DNA helicase is primarily responsible for This paper is available on line at http://www.jbc.org 22127 This is an Open Access article under the CC BY license. 22128 A Role for the TFIIH XPB DNA Helicase TFIIH action in suppression of arrest of early RNA polymerase II elongation complexes during their escape from the promoter. EXPERIMENTAL PROCEDURES Materials—Unlabeled ultrapure ribonucleoside 59-triphosphates, 39- O-MeGTP, and [a- P]CTP (.3000 Ci/mmol) were purchased from Am- ersham Pharmacia Biotech. Dinucleotides CpA and CpU, polyvinyl alcohol (type II) and a-amanitin were obtained from Sigma. Acetylated bovine serum albumin and recombinant human placental ribonuclease inhibitor were from Promega. Preparation of RNA Polymerase II and Transcription Factors—RNA polymerase II and TFIIH were purified from rat liver nuclear extracts as described (26). Recombinant yeast TBP (27, 28), recombinant TFIIB (29), and recombinant TFIIF (30) were expressed in Escherichia coli and purified as described previously (27–30). Recombinant TFIIE was prepared as described previously (31), except that the 56-kDa subunit was expressed in E. coli strain BL21(DE3)-pLysS. IIH6, IIH6/XPB- K346R, and IIH6/XPD-K48R were expressed in Sf9 cells and purified through the heparin Ultrogel chromatography step as described previ- ously (2). IIH6 and IIH6 mutants were further purified by anti-p44 immunoaffinity chromatography using the monoclonal antibody 1H5 (32). Recombinant CAK was purified as described previously (4). FIG.1. Recombinant IIH6, IIH6 mutants, and CAK. A, structure Assay of Transcription—Preinitiation complexes were assembled at of XPB and XPD. I–VI indicate the XPB and XPD helicase motifs. the AdML promoter on the EcoRI to NdeI fragment of pDN-AdML (33) K346R and K48R indicate the positions of point mutations in XPB and or on the premelted template fragment Ad(29/11) (18) at 28 °C by a XPD ATP binding sites, respectively. B, purified recombinant wild type 45– 60-min preincubation of 30-ml reaction mixtures containing 20 mM (lane 2) or mutant IIH6 (lanes 3 and 4) or TFIIH purified from HeLa Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 4 mM cells (heparin 5-PW fraction (41)) (lane 1) were separated by 12% MgCl , 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml bovine serum SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- albumin, 2% (w/v) polyvinyl alcohol, 3% (v/v) glycerol, 6 units of recom- bodies raised against each of the subunits. C, purified recombinant binant placental ribonuclease inhibitor, ;10 ng of DNA template frag- CAK was separated by 12% SDS-polyacrylamide gel electrophoresis ment, ;5 ng of recombinant TBP, ;10 ng of recombinant TFIIB, ;10 ng and immunoblotted with antibodies against CDK7, cyclin H (CycH), of recombinant TFIIF, ;20 ng of recombinant TFIIE, 0.01 unit of RNA and MAT1. polymerase II, and, where indicated, ;100 ng of CAK and either equiv- alent amounts (;150 ng) of wild type IIH6 or IIH6 mutants or ;10 ng as substrates for transcription, polymerase will efficiently syn- of rat TFIIH. Transcription was initiated by addition of 4 mlofa thesize abortively initiated, trinucleotide transcripts (10, 35, solution containing the nucleotides indicated in the figure legends. 36). We and others have shown previously that abortive initi- Reactions were stopped by addition of an equal volume of 9.0 M urea containing 0.025% (w/v) bromphenol blue and 0.025% (w/v) xylene ation from the AdML promoter can be measured in the pres- cyanol FF. Transcripts were analyzed by electrophoresis through poly- ence of [a- P]CTP and either initiating dinucleotide CpA or acrylamide gels containing 25% acrylamide, 3% bisacrylamide, 5.0 M CpU, which prime transcription at positions 21 and 23 rela- urea, 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA. Transcription tive to the normal AdML transcriptional start site (Fig. 2A) (35, was quantitated using a Molecular Dynamics PhosphorImager. 37, 38). In addition, we and others have shown that maximal RESULTS AND DISCUSSION rates of abortive initiation from the AdML promoter depend To investigate the roles of the XPB and XPD DNA helicases strongly on an ATP cofactor and all five general initiation and CAK in TFIIH-dependent promoter escape, we compared factors (21, 22, 38). the abilities of IIH6 and two IIH6 mutants, IIH6/XPB-K346R As shown in Fig. 2A, wild type IIH6 stimulated the rate of and IIH6/XPD-K48R, which contain point mutations in the abortive initiation above the low background level observed in XPB and XPD ATP binding sites and lack DNA helicase activ- the absence of TFIIH, whereas equivalent concentrations of the ity (24, 25), to suppress arrest of early RNA polymerase II XPB mutant IIH6/XPB-K346R and the XPD mutant IIH6/XPD- elongation intermediates in a minimal transcription system K48R did not. CAK, which is composed of CDK7, cyclin H, and reconstituted with purified polymerase and general initiation MAT1 subunits, detectably stimulated the rate of abortive ini- factors TBP, TFIIB, TFIIE, and TFIIF. IIH6 and IIH6 mutants tiation by both wild type IIH6 and the XPD mutant IIH6/XPD- were expressed in Sf9 cells coinfected with baculoviruses en- K48R, but not by the XPB mutant IIH6/XPB-K346R (Fig. 2B). coding human TFIIH subunits p34, p44, p52, p62, wild type or These findings are consistent with the results of Tirode et al. mutant XPD, and wild type or mutant XPB (2). Recombinant (2), who observed that the XPB mutant IIH6/XPB-K346R did IIH6 and IIH6 mutants were purified from lysates of Sf9 cells not support detectable open complex formation and runoff tran- by sequential heparin ultrogel and anti-p44 immunoaffinity scription in the presence or absence of CAK, whereas the XPD chromatography (2, 32). Recombinant CAK was purified from mutant IIH6/XPD-K48R was substantially less active than lysates of Sf9 cells coinfected with baculoviruses encoding IIH6, but could support a low level of runoff transcription that CDK7, cyclin H, and MAT1 (4). The subunit compositions of was stimulated by CAK. wild type and mutant IIH6 complexes and CAK were verified To investigate the activities of IIH6 and IIH6 mutants in by Western blotting, and the relative concentrations of wild promoter escape, we took advantage of the artificial AdML type and mutant IIH6 complexes were estimated by quantita- promoter derivative Ad(29/21), which contains a premelted tive Western blotting (Fig. 1 and data not shown). region from positions 29to 21 relative to the normal transcrip- To characterize the transcriptional activities of IIH6 and tional start site. The Ad(29/21) promoter supports transcrip- IIH6 mutants, we began by using a dinucleotide-primed abor- tion initiation by RNA polymerase II in the absence of TFIIH tive initiation assay to compare their abilities to support tran- and an ATP cofactor and is therefore a useful model for inves- scription initiation from the AdML promoter in the minimal tigating post-initiation roles of TFIIH and ATP (12, 16 –18, 39). transcription system. In the presence of an ATP cofactor, RNA We previously observed that maximal synthesis of 18 nucleo- polymerase II will utilize dinucleotides to prime synthesis of tide RNAs terminated at the first G residue of the Ad(29/21) promoter-specific transcripts (34). If only a dinucleotide primer transcript by incorporation of 39-O-MeG requires TFIIH and and the next nucleotide encoded by the template are provided ATP and is inhibited by ATPgS (18). Further elongation of the A Role for the TFIIH XPB DNA Helicase 22129 FIG.2. Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R in abortive initiation. RNA polymerase II preinitiation complexes were assembled at the AdML promoter as described under “Experimental Procedures.” Equivalent amounts of wild type IIH6 or IIH6 mutants (normalized to XPB polypeptide) and ;100 ng of CAK were added to reactions, as indicated in the figure, 30 min prior to addition of nucleotides. A, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for the times indicated in the figure in the presence of 170 mM CpA, 5 mM ATP, and 15 mCi of [a- P]CTP. B, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for 45 min in the presence of 170 mM CpA, 5 mM ATP, and 15 mCi of [a- P]CTP. Synthesis of trinucleotide transcripts was quantitated by PhosphorImager analysis. 18-nucleotide transcript is independent of ATP and TFIIH; thus, RNA polymerase II elongation complexes that have com- pleted synthesis of these transcripts can be considered to have escaped the promoter. To compare the abilities of IIH6 and IIH6 mutants to support efficient promoter escape, RNA polymerase II preinitiation complexes were assembled at the Ad(29/21) promoter in the minimal transcription system in the presence of either IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Transcription was car- ried out in the presence of ATP or ATPgS and the initiating dinucleotide CpU, UTP, [a- P]CTP, and 39-O-MeGTP. Reac- tion mixtures were then gel-filtered to remove unincorporated [a- P]CTP and the large number of abortive transcripts syn- thesized during transcription of premelted templates (40), see also Fig. 3C). As shown in Fig. 3B, in the presence of ATPgS, the majority of RNA polymerase II elongation intermediates suffered arrest before completing synthesis of the 18 nucleotide, 39-O-MeG- terminated transcript; similar levels of the 18-nucleotide tran- script were synthesized whether reactions contained IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Substitution of ATP for ATPgS increased accumulation of the 18-nucleotide transcript ;7-fold in reactions containing IIH6 and ;5-fold in reactions containing the XPD mutant IIH6/XPD-K48R. In contrast, sub- stitution of ATP for ATPgS had no significant effect on accu- mulation of the 18 nucleotide transcript in reactions containing the XPB mutant IIH6/XPB-K346R, arguing that the XPB DNA FIG.3. Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD- helicase makes a significantly greater contribution than the K48R in promoter escape. RNA polymerase II preinitiation com- XPD DNA helicase to TFIIH function in ATP-dependent pro- plexes were assembled at the premelted Ad(29/21) promoter as de- scribed under “Experimental Procedures.” Equivalent amounts of wild moter escape. type IIH6 and IIH6 mutants (normalized to XPB polypeptide) and ;100 As shown previously (2) and in Fig. 2B, the presence of the ng of CAK were added to reactions, as indicated in the figure, 30 min CAK subunits increases TFIIH activity in abortive initiation prior to addition of nucleotides. A, initial transcribed region of Ad(29/ and in synthesis of runoff transcripts. To investigate the con- 11) premelted promoter. B, synthesis of 18-nucleotide transcripts was tribution of CAK to TFIIH-dependent promoter escape, IIH6 carried out at 28 °C for 45 min in the presence of 170 mM CpU, 50 mM UTP, 50 mM 39-O-MeGTP, 15 mCi of [a- P]CTP, and either 50 mM ATP and IIH6 mutants were assayed in the presence and absence of or 50 mM ATPgS. Ternary elongation complexes were purified by cen- CAK, and reaction products were analyzed without prior gel trifugation through 300 ml of Sepharose CL-6B spin columns that had filtration. As shown in Fig. 3C, CAK had no detectable effect on been equilibrated in transcription buffer. Synthesis of 18-nucleotide the levels of 18 nucleotide transcripts synthesized in the pres- transcripts was quantitated by PhosphorImager analysis. C, synthesis of 18-nucleotide transcripts was carried out at 28 °C for 45 min in the ence of either wild type IIH6, IIH6/XPB-K346R, or IIH6/XPD- presence of 170 mM CpU, 50 mM UTP, 50 mM 39-O-MeGTP, 15 mCi of K48R, arguing that CAK does not contribute significantly to [a- P]CTP, and either 50 mM ATP or 50 mM ATPgS. TFIIH-dependent promoter escape. Because these reactions were not gel-filtered, a large number of abortive transcripts can 22130 A Role for the TFIIH XPB DNA Helicase Natl. Acad. Sci. U. S. A. 93, 6488 – 6493 be observed. Nonetheless, the relative amounts of 18 nucleotide 6. Reardon, J. T., Ge, H., Gibbs, E., Sancar, A., Hurwitz, J., and Pan, Z. Q. (1996) transcript synthesized in the presence of wild type IIH6, IIH6/ Proc. Natl. Acad. Sci. U. S. A. 93, 6482– 6487 7. Conaway, R. C., and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, XPB-K346R, and IIH6/XPD-K48R are comparable with those 7356 –7360 seen when reaction products were gel filtered prior to poly- 8. Conaway, R. C., and Conaway, J. W. (1993) Annu. Rev. Biochem. 62, 161–190 acrylamide gel electrophoresis (Fig. 3B, lanes 1, 3, and 5). 9. Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327–335 10. Wang, W., Carey, M., and Gralla, J. D. (1992) Science 255, 450 – 453 In summary, in this report we have taken advantage of 11. Jiang, Y., Smale, S. T., and Gralla, J. D. (1993) J. Biol. Chem. 268, 6535– 6540 recombinant TFIIH mutants to investigate the contributions of 12. Holstege, F. C. P., van der Vliet, P. C., and Timmers, H. Th. M. (1996) EMBO TFIIH DNA helicase and CTD kinase activities to efficient J. 15, 1666 –1677 13. Holstege, F. C. P., Fiedler, U., and Timmers, H. Th. M. (1997) EMBO J. 16, promoter escape by RNA polymerase II in a minimal transcrip- 7468 –7480 tion system reconstituted with purified polymerase and gen- 14. Parvin, J. D., and Sharp, P. A. (1993) Cell 73, 533–540 15. Parvin, J. D., Shykind, B. M., Meyers, R. E., Kim, J., and Sharp, P. A. (1994) eral initiation factors. By comparing the activities of the TFIIH J. Biol. Chem. 269, 18414 –18421 subassembly IIH6 and two IIH6 mutants, IIH6/XPB-K346R 16. Tantin, D., and Carey, M. (1994) J. Biol. Chem. 269, 17397–17400 and IIH6/XPD-K48R, which contain point mutations in the 17. Holstege, F., Tantin, D., Carey, M., van der Vliet, P. C., and Timmers, H. Th. M. (1995) EMBO J. 14, 810 – 819 XPB and XPD ATP binding sites and lack DNA helicase activ- 18. Dvir, A., Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad. Sci. ity (24, 25), we have obtained evidence supporting the model U. S. A. 94, 9006 –9010 that the XPB DNA helicase is primarily responsible for TFIIH 19. Yan, M., and Gralla, J. D. (1997) EMBO J. 16, 7457–7467 20. Conaway, J. W., Dvir, A., Moreland, R. J., Yan, Q., Elmendorf, B. J., Tan, S., action in ATP-dependent promoter escape. We observe (i) that and Conaway, R. C. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, the IIH6 point mutant IIH6/XPB-K346R, which contains wild 357–364 21. Kugal, J. F., and Goodrich, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, type XPD DNA helicase but lacks functional XPB DNA heli- 9232–9237 case, is inactive in promoter escape and (ii) that the IIH6 point 22. Kumar, K. P., Akoulitchev, S., and Reinberg, D. (1998) Proc. Natl. Acad. Sci. mutant IIH6/XPD-K48R, which contains wild type XPB DNA U. S. A. 95, 9767–9772 23. Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, helicase but lacks functional XPD DNA helicase, supports pro- 23352–23356 moter escape but less actively than wild type IIH6. Together 24. Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988) EMBO J. 7, 3263–3269 with the recent findings of Tirode et al. (2), who presented 25. Guzder, S. N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994) Nature evidence supporting the model (i) that the XPB DNA helicase is 369, 578 –581 essential for ATP-dependent formation of the open complex and 26. Conaway, R. C., Reines, D., Garrett, K. P., Powell, W., and Conaway, J. W. (1996) Methods Enzymol. 273, 194 –207 (ii) that the XPD DNA helicase stimulates this reaction, our 27. Schmidt, M. C., Kao, C. C., Pei, R., and Berk, A. J. (1989) Proc. Natl. Acad. Sci. results indicate that the relative contributions of the XPB and U. S. A. 86, 7785–7789 28. Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. 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A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 32, Issue of August 6, pp. 22127–22130, 1999 Communication © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. posed of the kinase/cyclin pair CDK7/cyclin H and the A Role for the TFIIH XPB DNA RING-H2 finger protein MAT1. TFIIH subunits are found in a Helicase in Promoter Escape variety of additional subassemblies, including a six-subunit complex (IIH6) containing XPB, XPD, p62, p52, p44, and p34, a by RNA Polymerase II* five-subunit “core” complex (IIH5) containing XPB, p62, p52, p44, and p34, and a four-subunit XPD/CAK complex (2– 6). (Received for publication, May 26, 1999) TFIIH was initially identified by its requirement in tran- Rodney J. Moreland‡, Franck Tirode§, scription initiation by RNA polymerase II (7). Initiation is an ¶ ¶i Qin Yan‡ , Joan Weliky Conaway‡ **, ATP-dependent process that requires at minimum the five Jean-Marc Egly§, and Ronald C. Conaway‡‡‡ general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and From the ‡Program in Molecular and Cell Biology, TFIIH (8, 9). Biochemical studies have shown that initiation in Oklahoma Medical Research Foundation, Oklahoma this minimal transcription system proceeds through multiple City, Oklahoma 73104, the §Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ stages beginning with assembly of polymerase and all five ULP, B. P. 163, Illkirch, C.U. de Strasbourg, France, general initiation factors into a closed preinitiation complex at the Department of Biochemistry and Molecular the promoter (8, 9) and culminating in ATP-dependent forma- Biology, University of Oklahoma Health Sciences tion of the open complex and synthesis of the first phosphodi- Center, Oklahoma City, Oklahoma 73190, and the ester bond of nascent transcripts (10 –13). Evidence supporting Howard Hughes Medical Institute, Oklahoma Medical a role for TFIIH DNA helicase activity in ATP-dependent for- Research Foundation, Oklahoma City, Oklahoma 73104 mation of the open complex was initially suggested by studies TFIIH is an RNA polymerase II transcription factor indicating that both TFIIH and ATP are dispensible for initi- that performs ATP-dependent functions in both tran- ation by RNA polymerase II from artificial promoters contain- scription initiation, where it catalyzes formation of the ing premelted transcriptional start sites and from promoters on open complex, and in promoter escape, where it sup- negatively supercoiled DNA templates (14 –19). presses arrest of the early elongation complex at pro- In addition to its requirement in transcription initiation, moter-proximal sites. TFIIH possesses three known ATP- TFIIH is also required for efficient promoter escape by RNA dependent activities: a 3*3 5* DNA helicase catalyzed by polymerase II (18, 20 –22). Mechanistic studies have shown its XPB subunit, a 5* 3 3* DNA helicase catalyzed by its that a fraction of early RNA polymerase II elongation interme- XPD subunit, and a carboxyl-terminal domain (CTD) ki- nase activity catalyzed by its CDK7 subunit. In this re- diates are prone to arrest at promoter-proximal sites in the port, we exploit TFIIH mutants to investigate the con- absence of TFIIH or an ATP cofactor (18, 21–23). Circumstan- tributions of TFIIH DNA helicase and CTD kinase tial evidence that TFIIH DNA helicase activity is responsible activities to efficient promoter escape by RNA polymer- for suppressing arrest of early elongation intermediates has ase II in a minimal transcription system reconstituted come from the observation that promoter escape is blocked by with purified polymerase and general initiation factors. the TFIIH DNA helicase inhibitor ATPgS, but not by the TFIIH Our findings argue that the TFIIH XPB DNA helicase is CTD kinase inhibitor H-8 (18). primarily responsible for preventing premature arrest Although evidence from previous studies suggested that of early elongation intermediates during exit of polym- TFIIH DNA helicase activity is required for ATP-dependent erase from the promoter. formation of the open complex and ATP-dependent promoter escape, a direct test of this hypothesis was not possible until sufficient quantities of purified TFIIH mutants lacking func- TFIIH is a nine-subunit complex that possesses multiple tional XPB or XPD DNA helicase were available. Recently, catalytic activities, including DNA-dependent ATPase, DNA some of us (F. Tirode and J.-M. Egly) reported the development helicase, and a protein kinase that is capable of phosphoryl- of methods for reconstitution of TFIIH and TFIIH subassem- ating the carboxyl-terminal domain (CTD) of the largest sub- blies from wild type and mutant subunits (2, 4). By investigat- unit of RNA polymerase II (1). The two largest TFIIH subunits ing the activities of TFIIH mutants, we observed that maximal are ATP-dependent DNA helicases encoded by the Xeroderma TFIIH transcriptional activity requires all nine subunits, al- pigmentosum complementation group B (XPB) and D (XPD) though the TFIIH subassembly IIH6 lacking CAK is active in genes. The TFIIH-associated CTD kinase is a three-subunit ATP-dependent formation of the open complex and supports a subassembly, CDK-activating kinase (CAK), which is com- reduced level of runoff transcription (4). In addition, by com- paring the activities of IIH6 and two IIH6 mutants, IIH6/XPB- * This work was supported in part by National Institutes of Health K346R and IIH6/XPD-K48R, which contain point mutations in Grant GM41628 (to R. C. C.), a Human Frontier Grant (to J. M. E.), and the XPB and XPD ATP binding sites and lack DNA helicase by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust. The costs of publi- activity (24, 25), we obtained evidence supporting the model cation of this article were defrayed in part by the payment of page that the XPB DNA helicase is essential for formation of the charges. This article must therefore be hereby marked “advertisement” open complex and runoff transcription and that the XPD DNA in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. helicase, though not essential, stimulates these reactions (2). ** Associate Investigator of the Howard Hughes Medical Institute. ‡‡ To whom correspondence should be addressed. Tel.: 405-271-1950; In this report, we exploit recombinant TFIIH mutants lack- Fax: 405-271-1580. ing functional XPB DNA helicase, XPD DNA helicase, or CAK The abbreviations used are: CTD, carboxyl-terminal domain; 39-O- to investigate the contribution of TFIIH DNA helicase and CTD MeGTP, 39-O-methylguanosine 59-triphosphate; ATPgS, adenosine 59- kinase activities to efficient promoter escape. Our findings O-(thio)triphosphate; AdML, adenovirus 2 major late; CAK, CDK-acti- vating kinase. argue that the XPB DNA helicase is primarily responsible for This paper is available on line at http://www.jbc.org 22127 This is an Open Access article under the CC BY license. 22128 A Role for the TFIIH XPB DNA Helicase TFIIH action in suppression of arrest of early RNA polymerase II elongation complexes during their escape from the promoter. EXPERIMENTAL PROCEDURES Materials—Unlabeled ultrapure ribonucleoside 59-triphosphates, 39- O-MeGTP, and [a- P]CTP (.3000 Ci/mmol) were purchased from Am- ersham Pharmacia Biotech. Dinucleotides CpA and CpU, polyvinyl alcohol (type II) and a-amanitin were obtained from Sigma. Acetylated bovine serum albumin and recombinant human placental ribonuclease inhibitor were from Promega. Preparation of RNA Polymerase II and Transcription Factors—RNA polymerase II and TFIIH were purified from rat liver nuclear extracts as described (26). Recombinant yeast TBP (27, 28), recombinant TFIIB (29), and recombinant TFIIF (30) were expressed in Escherichia coli and purified as described previously (27–30). Recombinant TFIIE was prepared as described previously (31), except that the 56-kDa subunit was expressed in E. coli strain BL21(DE3)-pLysS. IIH6, IIH6/XPB- K346R, and IIH6/XPD-K48R were expressed in Sf9 cells and purified through the heparin Ultrogel chromatography step as described previ- ously (2). IIH6 and IIH6 mutants were further purified by anti-p44 immunoaffinity chromatography using the monoclonal antibody 1H5 (32). Recombinant CAK was purified as described previously (4). FIG.1. Recombinant IIH6, IIH6 mutants, and CAK. A, structure Assay of Transcription—Preinitiation complexes were assembled at of XPB and XPD. I–VI indicate the XPB and XPD helicase motifs. the AdML promoter on the EcoRI to NdeI fragment of pDN-AdML (33) K346R and K48R indicate the positions of point mutations in XPB and or on the premelted template fragment Ad(29/11) (18) at 28 °C by a XPD ATP binding sites, respectively. B, purified recombinant wild type 45– 60-min preincubation of 30-ml reaction mixtures containing 20 mM (lane 2) or mutant IIH6 (lanes 3 and 4) or TFIIH purified from HeLa Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 4 mM cells (heparin 5-PW fraction (41)) (lane 1) were separated by 12% MgCl , 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml bovine serum SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- albumin, 2% (w/v) polyvinyl alcohol, 3% (v/v) glycerol, 6 units of recom- bodies raised against each of the subunits. C, purified recombinant binant placental ribonuclease inhibitor, ;10 ng of DNA template frag- CAK was separated by 12% SDS-polyacrylamide gel electrophoresis ment, ;5 ng of recombinant TBP, ;10 ng of recombinant TFIIB, ;10 ng and immunoblotted with antibodies against CDK7, cyclin H (CycH), of recombinant TFIIF, ;20 ng of recombinant TFIIE, 0.01 unit of RNA and MAT1. polymerase II, and, where indicated, ;100 ng of CAK and either equiv- alent amounts (;150 ng) of wild type IIH6 or IIH6 mutants or ;10 ng as substrates for transcription, polymerase will efficiently syn- of rat TFIIH. Transcription was initiated by addition of 4 mlofa thesize abortively initiated, trinucleotide transcripts (10, 35, solution containing the nucleotides indicated in the figure legends. 36). We and others have shown previously that abortive initi- Reactions were stopped by addition of an equal volume of 9.0 M urea containing 0.025% (w/v) bromphenol blue and 0.025% (w/v) xylene ation from the AdML promoter can be measured in the pres- cyanol FF. Transcripts were analyzed by electrophoresis through poly- ence of [a- P]CTP and either initiating dinucleotide CpA or acrylamide gels containing 25% acrylamide, 3% bisacrylamide, 5.0 M CpU, which prime transcription at positions 21 and 23 rela- urea, 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA. Transcription tive to the normal AdML transcriptional start site (Fig. 2A) (35, was quantitated using a Molecular Dynamics PhosphorImager. 37, 38). In addition, we and others have shown that maximal RESULTS AND DISCUSSION rates of abortive initiation from the AdML promoter depend To investigate the roles of the XPB and XPD DNA helicases strongly on an ATP cofactor and all five general initiation and CAK in TFIIH-dependent promoter escape, we compared factors (21, 22, 38). the abilities of IIH6 and two IIH6 mutants, IIH6/XPB-K346R As shown in Fig. 2A, wild type IIH6 stimulated the rate of and IIH6/XPD-K48R, which contain point mutations in the abortive initiation above the low background level observed in XPB and XPD ATP binding sites and lack DNA helicase activ- the absence of TFIIH, whereas equivalent concentrations of the ity (24, 25), to suppress arrest of early RNA polymerase II XPB mutant IIH6/XPB-K346R and the XPD mutant IIH6/XPD- elongation intermediates in a minimal transcription system K48R did not. CAK, which is composed of CDK7, cyclin H, and reconstituted with purified polymerase and general initiation MAT1 subunits, detectably stimulated the rate of abortive ini- factors TBP, TFIIB, TFIIE, and TFIIF. IIH6 and IIH6 mutants tiation by both wild type IIH6 and the XPD mutant IIH6/XPD- were expressed in Sf9 cells coinfected with baculoviruses en- K48R, but not by the XPB mutant IIH6/XPB-K346R (Fig. 2B). coding human TFIIH subunits p34, p44, p52, p62, wild type or These findings are consistent with the results of Tirode et al. mutant XPD, and wild type or mutant XPB (2). Recombinant (2), who observed that the XPB mutant IIH6/XPB-K346R did IIH6 and IIH6 mutants were purified from lysates of Sf9 cells not support detectable open complex formation and runoff tran- by sequential heparin ultrogel and anti-p44 immunoaffinity scription in the presence or absence of CAK, whereas the XPD chromatography (2, 32). Recombinant CAK was purified from mutant IIH6/XPD-K48R was substantially less active than lysates of Sf9 cells coinfected with baculoviruses encoding IIH6, but could support a low level of runoff transcription that CDK7, cyclin H, and MAT1 (4). The subunit compositions of was stimulated by CAK. wild type and mutant IIH6 complexes and CAK were verified To investigate the activities of IIH6 and IIH6 mutants in by Western blotting, and the relative concentrations of wild promoter escape, we took advantage of the artificial AdML type and mutant IIH6 complexes were estimated by quantita- promoter derivative Ad(29/21), which contains a premelted tive Western blotting (Fig. 1 and data not shown). region from positions 29to 21 relative to the normal transcrip- To characterize the transcriptional activities of IIH6 and tional start site. The Ad(29/21) promoter supports transcrip- IIH6 mutants, we began by using a dinucleotide-primed abor- tion initiation by RNA polymerase II in the absence of TFIIH tive initiation assay to compare their abilities to support tran- and an ATP cofactor and is therefore a useful model for inves- scription initiation from the AdML promoter in the minimal tigating post-initiation roles of TFIIH and ATP (12, 16 –18, 39). transcription system. In the presence of an ATP cofactor, RNA We previously observed that maximal synthesis of 18 nucleo- polymerase II will utilize dinucleotides to prime synthesis of tide RNAs terminated at the first G residue of the Ad(29/21) promoter-specific transcripts (34). If only a dinucleotide primer transcript by incorporation of 39-O-MeG requires TFIIH and and the next nucleotide encoded by the template are provided ATP and is inhibited by ATPgS (18). Further elongation of the A Role for the TFIIH XPB DNA Helicase 22129 FIG.2. Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R in abortive initiation. RNA polymerase II preinitiation complexes were assembled at the AdML promoter as described under “Experimental Procedures.” Equivalent amounts of wild type IIH6 or IIH6 mutants (normalized to XPB polypeptide) and ;100 ng of CAK were added to reactions, as indicated in the figure, 30 min prior to addition of nucleotides. A, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for the times indicated in the figure in the presence of 170 mM CpA, 5 mM ATP, and 15 mCi of [a- P]CTP. B, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for 45 min in the presence of 170 mM CpA, 5 mM ATP, and 15 mCi of [a- P]CTP. Synthesis of trinucleotide transcripts was quantitated by PhosphorImager analysis. 18-nucleotide transcript is independent of ATP and TFIIH; thus, RNA polymerase II elongation complexes that have com- pleted synthesis of these transcripts can be considered to have escaped the promoter. To compare the abilities of IIH6 and IIH6 mutants to support efficient promoter escape, RNA polymerase II preinitiation complexes were assembled at the Ad(29/21) promoter in the minimal transcription system in the presence of either IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Transcription was car- ried out in the presence of ATP or ATPgS and the initiating dinucleotide CpU, UTP, [a- P]CTP, and 39-O-MeGTP. Reac- tion mixtures were then gel-filtered to remove unincorporated [a- P]CTP and the large number of abortive transcripts syn- thesized during transcription of premelted templates (40), see also Fig. 3C). As shown in Fig. 3B, in the presence of ATPgS, the majority of RNA polymerase II elongation intermediates suffered arrest before completing synthesis of the 18 nucleotide, 39-O-MeG- terminated transcript; similar levels of the 18-nucleotide tran- script were synthesized whether reactions contained IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Substitution of ATP for ATPgS increased accumulation of the 18-nucleotide transcript ;7-fold in reactions containing IIH6 and ;5-fold in reactions containing the XPD mutant IIH6/XPD-K48R. In contrast, sub- stitution of ATP for ATPgS had no significant effect on accu- mulation of the 18 nucleotide transcript in reactions containing the XPB mutant IIH6/XPB-K346R, arguing that the XPB DNA FIG.3. Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD- helicase makes a significantly greater contribution than the K48R in promoter escape. RNA polymerase II preinitiation com- XPD DNA helicase to TFIIH function in ATP-dependent pro- plexes were assembled at the premelted Ad(29/21) promoter as de- scribed under “Experimental Procedures.” Equivalent amounts of wild moter escape. type IIH6 and IIH6 mutants (normalized to XPB polypeptide) and ;100 As shown previously (2) and in Fig. 2B, the presence of the ng of CAK were added to reactions, as indicated in the figure, 30 min CAK subunits increases TFIIH activity in abortive initiation prior to addition of nucleotides. A, initial transcribed region of Ad(29/ and in synthesis of runoff transcripts. To investigate the con- 11) premelted promoter. B, synthesis of 18-nucleotide transcripts was tribution of CAK to TFIIH-dependent promoter escape, IIH6 carried out at 28 °C for 45 min in the presence of 170 mM CpU, 50 mM UTP, 50 mM 39-O-MeGTP, 15 mCi of [a- P]CTP, and either 50 mM ATP and IIH6 mutants were assayed in the presence and absence of or 50 mM ATPgS. Ternary elongation complexes were purified by cen- CAK, and reaction products were analyzed without prior gel trifugation through 300 ml of Sepharose CL-6B spin columns that had filtration. As shown in Fig. 3C, CAK had no detectable effect on been equilibrated in transcription buffer. Synthesis of 18-nucleotide the levels of 18 nucleotide transcripts synthesized in the pres- transcripts was quantitated by PhosphorImager analysis. C, synthesis of 18-nucleotide transcripts was carried out at 28 °C for 45 min in the ence of either wild type IIH6, IIH6/XPB-K346R, or IIH6/XPD- presence of 170 mM CpU, 50 mM UTP, 50 mM 39-O-MeGTP, 15 mCi of K48R, arguing that CAK does not contribute significantly to [a- P]CTP, and either 50 mM ATP or 50 mM ATPgS. TFIIH-dependent promoter escape. Because these reactions were not gel-filtered, a large number of abortive transcripts can 22130 A Role for the TFIIH XPB DNA Helicase Natl. Acad. Sci. U. S. A. 93, 6488 – 6493 be observed. Nonetheless, the relative amounts of 18 nucleotide 6. Reardon, J. T., Ge, H., Gibbs, E., Sancar, A., Hurwitz, J., and Pan, Z. Q. (1996) transcript synthesized in the presence of wild type IIH6, IIH6/ Proc. Natl. Acad. Sci. U. S. A. 93, 6482– 6487 7. Conaway, R. C., and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, XPB-K346R, and IIH6/XPD-K48R are comparable with those 7356 –7360 seen when reaction products were gel filtered prior to poly- 8. Conaway, R. C., and Conaway, J. W. (1993) Annu. Rev. Biochem. 62, 161–190 acrylamide gel electrophoresis (Fig. 3B, lanes 1, 3, and 5). 9. Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327–335 10. Wang, W., Carey, M., and Gralla, J. D. (1992) Science 255, 450 – 453 In summary, in this report we have taken advantage of 11. Jiang, Y., Smale, S. T., and Gralla, J. D. (1993) J. Biol. Chem. 268, 6535– 6540 recombinant TFIIH mutants to investigate the contributions of 12. Holstege, F. C. P., van der Vliet, P. C., and Timmers, H. Th. M. 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Sci. ity (24, 25), we have obtained evidence supporting the model U. S. A. 94, 9006 –9010 that the XPB DNA helicase is primarily responsible for TFIIH 19. Yan, M., and Gralla, J. D. (1997) EMBO J. 16, 7457–7467 20. Conaway, J. W., Dvir, A., Moreland, R. J., Yan, Q., Elmendorf, B. J., Tan, S., action in ATP-dependent promoter escape. We observe (i) that and Conaway, R. C. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, the IIH6 point mutant IIH6/XPB-K346R, which contains wild 357–364 21. Kugal, J. F., and Goodrich, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, type XPD DNA helicase but lacks functional XPB DNA heli- 9232–9237 case, is inactive in promoter escape and (ii) that the IIH6 point 22. Kumar, K. P., Akoulitchev, S., and Reinberg, D. (1998) Proc. Natl. Acad. Sci. mutant IIH6/XPD-K48R, which contains wild type XPB DNA U. S. A. 95, 9767–9772 23. Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, helicase but lacks functional XPD DNA helicase, supports pro- 23352–23356 moter escape but less actively than wild type IIH6. Together 24. Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988) EMBO J. 7, 3263–3269 with the recent findings of Tirode et al. (2), who presented 25. Guzder, S. N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994) Nature evidence supporting the model (i) that the XPB DNA helicase is 369, 578 –581 essential for ATP-dependent formation of the open complex and 26. Conaway, R. C., Reines, D., Garrett, K. P., Powell, W., and Conaway, J. W. (1996) Methods Enzymol. 273, 194 –207 (ii) that the XPD DNA helicase stimulates this reaction, our 27. Schmidt, M. C., Kao, C. C., Pei, R., and Berk, A. J. (1989) Proc. Natl. Acad. Sci. results indicate that the relative contributions of the XPB and U. S. A. 86, 7785–7789 28. Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. 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