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Efficient trans‐cleavage of a stem‐loop RNA substrate by a ribozyme derived from neurospora VS RNA.

Efficient trans‐cleavage of a stem‐loop RNA substrate by a ribozyme derived from neurospora VS RNA. EMBO Journal vol.14 no.2 1995 The pp.368-376, of a stem RNA Efficient trans-cleavage -loop a derived from substrate by ribozyme Neurospora VS RNA et al., 1990) and HDV (Branch and Robertson, 1991; C.T.Guo and Richard A.Collins1 Hans Perrotta and Been, 1992; Wu et al., 1992) ribozymes Canadian Instutute for Advanced Research Program in Evolutionary have been facilitated by altering these RNAs to perform Biology, Department of Molecular and Medical Genetics, University of intermolecular trans-cleavage reactions. In a trans- Canada M5S IA8 Toronto, Toronto, Ontario, cleavage reaction one RNA, the substrate, contains the author 'Corresponding site to be cleaved; a separate RNA, the ribozyme, provides Communicated by T.R.Cech the to the One sequences required catalyze cleavage. has been naturally occurring trans-acting ribozyme a 144 We have constructed ribozyme containing the RNA of RNase which discovered, component P, VS RNA that can nucleotides of Neurospora catalyze in trans cleaves pre-tRNA precursors (Guerrier-Takada of a RNA in a true the cleavage separate enzymatic reactions of most et al., 1983). Trans-cleavage ribozymes manner 0.13 0.7/min). have been such that of the substrate (Km ,uM, kcat Comparison designed binding of the rates of cis- and as well as the trans-cleavage, occurs via formation of Watson-Crick base multiple lack of effect of pH on the rate of cleavage, suggest with the Non-Watson-Crick and pairs ribozyme. tertiary that a rate-limiting step, possibly conformational interactions are also involved in substrate and binding The minimum con- change, occurs prior to cleavage. for et may be essential proper binding (Pyle al., 1992; for con- tiguous substrate sequence required cleavage et Guerrier-Takada Smith et al., 1992; Dib-Haij al., 1993; and 19 nucleotides sists of one nucleotide upstream and Altman, 1993). the site. Unlike most other downstream of cleavage and I it With hammerhead, hairpin Group ribozymes which interact with ribozymes long single-stranded nucleotides in the has been found that very few specific the minimal substrate for regions of their substrates, that substrate are required for trans-cleavage, provided consists of a stable the VS ribozyme mostly stem-loop, the adjacent region(s) are complementary to the binding which would to its appear preclude recognition simply the site on the ribozyme. This property has allowed via extensive Watson-Crick base pairing. engineering of ribozymes that can cleave sequences other Key words: cleavage/Neurospora/plasmid/RNA/ribozyme than those recognized by the naturally occurring ribozyme. Some also function in vivo in non- engineered ribozymes native host cells, which has raised the possibility of their use as therapeutic agents in dominant inherited disorders Introduction and against retroviruses and RNA viruses (reviewed by Castanotto et al., 1992). A small number of RNAs isolated from a of variety We are the VS RNA which is found in studying catalytic natural sources have been found to a possess self-cleavage the mitochondria of certain natural isolates of Neurospora that is involved in multimeric tran- activity processing and VS the (Collins Saville, 1990). performs same type scripts into monomers, apparently as part of the replication of RNA as cleavage hammerhead, hairpin and HDV cycle. Several different RNA sequences and secondary ribozymes, leaving products with 2',3'-cyclic phosphate structures appear to be capable of such activity. These and 5'-OH termini and include: in (Saville Collins, 1990), but it the hammerhead, found several plant viral is different in sequence, secondary structure, choice of satellite a RNAs, viroid RNA and the transcript of a cleavage site and functional properties from these other nuclear satellite DNA of a newt (Forster and Symons, ribozymes (Collins and Olive, 1993; Guo et al., 1993; 1987; reviewed by Symons, 1992); the hairpin (or paper T.L.Beattie, J.E.Olive and R.A.Collins, submitted). An clip) in the minus strand of the satellite of tobacco ringspot understanding of the structure and function of VS RNA virus and related viruses (Buzayan et al., 1986; Feldstein may therefore provide unique information about the ways et al., 1990; Hampel et al., 1990; Feldstein and Breuning, 1993); the genomic and antigenomic RNAs of hepatitis that RNAs can catalyze reactions and about the nature of delta virus (HDV; Kuo et al., 1988; Sharmeen et al., 1988; RNA active sites. Perrotta and Been, 1991, 1993); and VS RNA in the We report here that the catalytic region of VS RNA mitochondria of certain Neurospora isolates (Saville and can cleave a separate substrate RNA at a specific target Collins, 1990). site. The minimal substrate forms a stable hairpin with In their natural contexts the ribozymes mentioned above, few and only short single-stranded regions potentially as well as others such as Group I (Cech, 1990) and Group available for recognition by the ribozyme via Watson- II introns (Michel et al., 1989), perform intramolecular Crick pairing. Nonetheless, the low Km of the VS cis-cleavage and, in some cases, ligation reactions. trans-cleavage reaction (0.13 ,uM) suggests that the VS Structure-function studies of Group I introns (Szostak, ribozyme binds its substrate quite efficiently, implying 1986; Zaug et al., 1986) and later hammerhead that substrate recognition involves multiple, including (Uhlenbeck, 1987), hairpin (Feldstein et al., 1990; Hampel tertiary, interactions. 38 Oxford University Press Trans-cleavage by a VS RNA ribozyme Results of Gl 1/Ava substrate (S) A. sequence VS RNA can cleave a separate RNA substrate in gggaaagcuUGCGAAGGGCGUCGUCGCCCCG A trans 5 A 3, The trans reaction described below was constructed before cleavage site we had determined the secondary structure of VS RNA. In the absence of structure-based predictions about where B. to divide the normally cis-cleaving RNA to establish a trans-cleaving system, we used several restriction frag- S (32 nt) 3' ments of VS DNA cloned in a T7 promoter vector to + Ribozyme (Rz) construct pairs of non-overlapping regions of VS RNA. One member of each pair, the substrate (S; see Materials PI (13 nt) and methods), contained the expected cleavage site, fol- P2 (19 nt) lowing nucleotide G620 (numbered as in Saville and Collins, 1990); the other, the enzyme or ribozyme (Rz; RNA s S + Rz see Materials and methods), contained the remainder of the VS sequence, terminating at the SspI site at nucleotide time (min) 0 1 2 4 6 101530 60 783. In preliminary experiments (Saville, 1991; Guo, 1992) these transcripts were mixed at an 1:1 ratio and &&A A incubated under conditions known to support cis-cleavage (Collins and Olive, 1993). Most combinations showed little or no cleavage, however, almost complete cleavage of a 32 nucleotide substrate RNA that terminates at the AvaI site (nucleotide 639) was observed during a 1 h incubation with a ribozyme that begins at the AvaI site P2 ~ u4 _ and ends at the SspI site (nucleotide 783); no cleavage was observed in the absence of ribozyme (Figure 1). The electrophoretic mobilities of the two cleavage products, P1 ~ o were approximately those expected for cleavage P1 and P2, (confirmed below), which is the site after nucleotide 620 cis-cleavage of VS RNA. We chose to of intramolecular Fig. 1. The trans-cleavage reaction. (A) The nucleotide sequence of the GI l/AvaI substrate RNA (S) transcribed from clone GIl which this trans-cleavage reaction in further detail. examine with AvaI. VS nucleotides 617-639 had been linearized by digestion and Collins, 1990) are indicated in upper case; (numbered as in Saville are in lower case. (B) Time course of trans- vector nucleotides occurs at the same site as cis- Trans-cleavage (Rz). 32P-Labeled S and unlabeled cleavage of S by the AvaI ribozyme cleavage were incubated in 50 mM Tris-HCl, pH 7.5, Rz (0.05 jiM each) determine the precise site of cleavage, S, P1 and P2 To at 37°C. Aliquots were removed at the times indicated 20 mM MgC12 at their 5' ends and sequenced by partial were labeled electrophoresis and autoradiography (see Materials and analyzed by for details). The left lane contains a control incubation in using RNases TI or U2 (Figure 2). and methods enzymatic digestion of Rz. P1 and P2 are the cleavage products of S. the absence of a mutant substrate containing a Cleavage products substitution 3' of the cleavage site (A621U) single base between nucleotides 620 and 621, as in the cis- to resolve possible ambiguities occurred were also characterized reaction. to anomalous migration of some bands (see below). cleavage due and P1 are identical in sequence from the 5' Because S Minimal length of the substrate RNA the site, all RNase sequencing bands co- end to cleavage downstream To determine the minimal sequence required as (Figure 2A-C). Full-length P1 co- migrated, expected we used essentially the approach of the cleavage site, with the 13 nucleotide RNase TI fragment of S migrated and (1987) and used for described by Forster Symons that terminates at G620, which is the site of intramolecular et al. Perotta and Been, other riboyzmes (Feldstein 1989; in VS RNA. Also, the 3' end of P1 was cis-cleavage RNA was partially 1992). 5'-End-labeled G11/SspI to be 2',3'-cyclic phosphate (Figure 2F), found guanosine treatment at high pH, then incubated with location and chemical pathway of hydrolyzed by that both the indicating 3A). Incubation in the same as in the reaction. or without the ribozyme (Figure are the cis-cleavage trans-cleavage confirmed our 2) previous of a at absence of ribozyme (lane As from the finding cyclic phosphate expected Gi RNA and deletion was found at the that full-length 1/SspI end of a 5' group finding the 3' P1, hydroxyl < 10 nucleotides at the 3' end can cis- evidenced its derivatives lacking 5' end of as by end-labeling by P2, Incubation with the ribozyme et 1993). T4 kinase without prior cleave (Guo al., and polynucleotide [,y-32P]ATP or at least decrease in resulted in the disappearance, Alkaline ladders of 5' treatment. hydrolysis phosphatase to RNAs terminating at of bands corresponding P2 contained 18 of the expected 19 intensity, end-labeled only A few RNAs were not or nucleotide 639 longer (lane 4). This is the result of a artefact involving bands. compression these under conditions, indicating cleaved to completion of a stable stem-loop structure in the the formation very substrates. The minimal are poorer that they relatively this will be described in detail below. longer RNAs; terminates at residue which by substrate 639, 5' terminal nucleotides of P2 derived length the Nonetheless, to the RNA used in precisely S and the A621U mutant were A and U coincidence corresponds of from cleavage run-off was transcription which by 2G and that cleavage Figure 1, synthesized H), confirming respectively (Figure 369 and R.A.Collins H.C.T.Guo E. D. A. B. C. P2 G 1 P2 621U Gll S G1 Pl 621U P1 1 23A45 1 23 A45 12 3A45 12345 12345 G638 G638 G630 G633 -G62<8 G618 G625 G624 a * a , G623 9 eM *. F. G. H. Ribonuclease TI and U2 of 5' end-labeled S and of the sequencing cleavage products P1 Fig. 2. Characterization trans-cleavage products. (A-E) or incubated in TI or U2 buffer without RNases lanes clone GIl and mutant 621U. Lanes 1-3 are controls respectively); A, and P2 from (untreated RNase lanes RNase U2. Nucleotide are indicated to the left of the VS nucleotides alkaline lanes TI; 5, sequences figure. partial hydrolysis ladder; 4, in the site of is indicated the arrowhead. Thin of: the are in vector nucleotides lower cleavage by (F-H) layer chromatography (F) upper case, case; of 5' GIl the 5' nucleotide of 5' 621U P2. 3' nucleotide of GIl P1; (G) the 5' nucleotide phosphorylated P2; (H) phosphorylated of the 1 linearized at the AvaI site. Thus 19 characterization trans-cleavage products (Figures of a template only that were consistent nucleotides downstream of the cleavage site are required and 2) we noted several observations P2 than expected for the AvaI with such a structure. migrated faster trans-cleavage by ribozyme. RNA relative to size markers for a 19 nucleotide RNA, sug- A experiment using 3' end-labeled showed parallel of the site that only a single nucleotide upstream cleavage gesting that it contained a structure that was not fully for Taken together denatured even in a gel containing 8.3 M urea (Figure 1 is required trans-cleavage (Figure 3B). with the results from 5' end-labeled RNA 3A), and data not shown). Certain guanosine (623-625, 627 (Figure these data show that the minimum contiguous region of and and adenosine and 622) residues in S and 633) (621 the native RNA required for trans-cleavage consists of P2 were cleaved weakly or not at all by RNases TI and/ one nucleotide of the and 19 upstream cleavage site or U2, even though sequencing reactions were performed nucleotides downstream. under putatively denaturing conditions of 50°C, 1 mM EDTA and 7 M urea (Figure 2A-E). Only 18 of the The minimal substrate RNA consists mostly of a expected 19 bands were observed in the 5' end-labeled stable hairpin loop alkaline hydrolysis products of P2 (in the gel shown partial RNA structure prediction using the MFOLD program of in Figure 2D and E, the first nucleotide, A621, has been Zuker and collaborators (Zuker, 1989; data not shown) run off the gel, so only 17 bands are shown). Also, the suggests that the most thermodynamically reasonable spacing and intensity of bands at C634-C636 was uneven structure of the substrate RNA would be the hairpin- (Figure 2D and E), typical of compression artefacts during containing structure drawn in Figure 4B. During the caused by a secondary structure in the electrophoresis 370 a Trans-cleavage by VS RNA ribozyme A. cleavage B. cleavage site B.site G620 U783 G620 A639 52 p _* _ 5t- 3T 35$cp partial alkaline partial partial partial hydrolysis parl partial alkalineT Ti hydrolysi's digestion +Rz digestion -Rz -Rz +Rz markers 60' 0' 60' lane: i 2 3 C D 1' 30' 60' E -O 60' I'll / / 4' \// cleavable RNAs G61 8 G620 " I -w- P2 cleavE,able RNAs 5;; ... :-7 G630 ; +- * * .9 .- F , * _ G640- _ G638- g * '* *4 * .-NW q~ -__ G620- -n P1 G638 * * .23 t? 1 2 3 4 5 6 7 Fig. 3. Minimal substrate requirements for trans-cleavage of VS substrates by the AvaI ribozyme. End-labeled substrate RNAs, diagrammed at the top of each panel, were treated as indicated in the flow charts. (A) 5'-End-labeled GI I/SspI substrate. Substrate (0.3 was incubated with Rz gM) (0.3 in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 at 37°C for 0 or 60 min. (B) gM) 3'-[32P]pCp-End-labeled GI I/AvaI substrate. Substrate (0.05 and Rz (1.5 FM) were incubated as in (A) and samples were withdrawn at the gM) times indicated in lanes 4-7. To facilitate interpretation, relevant guanosines are indicated in the TI marker lane. P1 and P2 mark the position of the detectable (end-labeled) cleavage product in (A) and (B) respectively. longer molecules. The pattern and intensity of bands is although T1, G620 itself was only weakly cleaved. The consistent with the interpretation that the two RNAs in only strong TI cleavages downstream of G620 were at the alkaline ladder that terminate at C635 and C636 G630 in the were loop and G638 in the 3' single-stranded compressed; the RNA terminating at C635 is denatured, region. Guanosines in the stem were resistant to TI like those shorter than it (below it in the gel), while the cleavage. The RNase TI data are not consistent with an RNA ending at C636 extends the putative helix by one alternative structure in which two substrate RNAs dimerize base pair, thereby preventing its denaturation under to form a partially self-complementary duplex. In such these electrophoretic conditions. The duplex, G630 hairpin-containing would be paired and inaccessible to RNase RNA co-migrates with the but shorter, denatured, product TI. RNase T2, which has specificity for single-stranded terminating at C635. regions (Knapp, 1989), cleaved in the loop (U628, C629) results were Complementary obtained from RNase and at some positions upstream of the stem (U617, C619) sequencing of 3' end-labeled S and P2 (data not and weakly at A622. shown), A621, The weak TI and T2 cleavages i.e. the same guanosines and adenosines were resistant at to several positions (620-622) adjacent to the Watson- cleavage by the RNases and the alkaline Crick hydrolysis ladder base pairs in the hairpin stem may indicate that had a compression the two RNAs with 5' some structure involving extends beyond that drawn in Figure 4B. ends at G623 and G624 to C636 (those complementary and C635). Reaction conditions Nuclease under probing native conditions is also con- We have the effects of several variables investigated that sistent with the formation of a stable in S would be expected to affect RNA structure hairpin loop and that have (Figure 4A and data not Guanosines of been shown). found to affect the cleavage rates of other upstream ribozymes. the site at G620 to be We ribozyme cleavage arbitrarily chose an ratio of S and appeared single- equimolar Rz as were to RNase (0.05 stranded, they susceptible iM each) for most initial cleavage by investigations; more 371 H.C.T.Guo and R.A.Collins A. B. A. cu RNase RNase Ti T2 .' 100 oJ S 10 u C 1I 0 20 40 60 0 20 40 60 80 stem 3' mM temp (C) [MgCI2] G633 loop G630 C. D. 100 10Q0 NaCI G627 2E 100' stem 5' KCI E 10 - G625 G624 * S do G623 0 0 .1 .2 .4 2 4 6 8 10 .3 .5 G620 4*0 M [spermidine] mM [monovalent] ip. Ga618 *Q F. 100 'pp.4 100 E 10 \ E,g 7 7.5 8.5 9 0 2 8 4 6 8 10 mM pH [MgCI2] ¶4 5. Effect of Fig. reaction conditions on cleavage of S by Rz. Unless (A-E) otherwise indicated, reactions were carried out as B. Minimal secondary structure of Gl 1/Ava S described in Materials and methods at 37°C in the presence of 50 mM Tris-HCI, 10 mM pH 7.5, MgCl2 and the variables indicated on the 0 L 0 c')v X axes. The effect (F) of [MgCI2] under otherwise 'optimized' CQN NNI D Ut (D CO to to conditions: 50 mM 30°C, Tris-HCI, pH 8.0, 25 mM KC1, 2 mM 5,gggaaagcuUGC spermidine. GMGGGCG cleavage site AGCCCCGC UG3 detailed analyses specifically under either steady-state or co S single turnover conditions are described later. Cleavage rate increased with temperature until an optimum was around reached 30°C and then decreased C. sharply above 40°C (Figure No reaction 5A). was observed . .. ii 11 11 in the absence of a divalent cation and reaction rate increased with increasing MgCl2, reaching a maximum around 100 mM when magnesium was the only cation I present (Figure 5B). To determine whether some of 620 the MgCl2 was as a structural acting simply the counterion, effects of spermidine, NaCl, and KCI were investigated III 1IIl III I II 111111111) 5/ in the presence of a subsaturating concentration of MgCl2 5 3' \ (10 mM). In the presence of 10 mM MgCl2, spermidine at 1 mM or greater enhanced the rate of cleavage nearly 10- fold compared with the same reaction without spermidine 4. Structure of S and Rz (Figure 5C). Low concentrations of Fig. RNAs. (A) Limited RNase TI and T2 KCI (<100 mM) also digestion. 5'-End-labeled S was partially digested with RNase TI or stimulated the reaction rate to up -10-fold. Perhaps T2 at 30°C in native buffer (50 mM Tris-HCI, pH 8.0, 25 mM KCI, surprisingly, NaCl had almost no effect (Figure 5D). These 10 mM MgCl2, 2 mM spermidine) for 0.5-30 min. A guanosine observations are similar to the effects of cations observed marker lane (labeled TI denatured) was Ti prepared by RNase previously on the rate of in cis-cleavage of VS RNA sequencing denaturing buffer (see Materials and methods for (Collins details). Controls 1 and 2 contain RNA incubated in the absence of and The Olive, 1993). stimulation by low concentrations RNase in denaturing buffer or native buffer respectively. OH indicates of some other cations suggests that some MgCl2 was partial alkaline hydrolysis ladder. Nucleotides contributing to the acting as a counterion which could be replaced more stem-loop structure are indicated on the right. (B) Minimal secondary effectively by spermidine or potassium. structure of S. VS are in nucleotides upper case, vector in nucleotides lower case. The box indicates the The rate of minimal length of the substrate reaction showed only a small pH dependence. determined from deletion experiments shown in Figure 3. The nearly 100-fold increase in the hydroxide concentra- (C) Proposed secondary structure of the minimal cis-cleaving region tion between pH 7.1 and 8.9 resulted in only a 2-fold of VS RNA (T.L.Beattie, J.E.Olive and R.A.Collins, submitted). The increase in rate (Figure 5E). The effect of pH specifically box encloses the minimal substrate for the trans reaction (boxed in B). Rz consists of the under single turnover sequences downstream of the conditions is described later. substrate, forming stems II-VI (see Materials and methods). Finally, the effect of MgCl2 was re-assayed under 372 Trans-cleavage by a VS RNA ribozyme contrast, we observed that cleavage continued at a constant A. B. rate until -40% of S was cleaved and then decreased 1.5 slowly as the concentration of available uncleaved S I- decreased. This indicated that Rz behaved like a true l_ A enzyme in that it was capable of multiple rounds of v0 cleavage. Also, as expected of an enzyme, the initial rate 0.6 EL of cleavage was directly proportional to the concentration :0-3 of the ribozyme under conditions of substrate excess z0. (Figure 7B). 0.1II 01- . . . . . . The trans-cleavage reaction exhibits a saturation curve 0 1 2 3 4 5 10 6.0 7.0 8.0 9.0 with respect to substrate concentration that is typical of uM pH [Rz] Michaelis-Menten kinetics (Figure 7C). A Km of 0.13 jM Fig. 6. Effects of pH on the rate of trans-cleavage under single and kcat of 0.7/min were obtained from these data. These turnover conditions. (A) Determining single turnover conditions. Initial values have been observed to vary by a factor of -2 rates of cleavage of S (0.13 by increasing concentrations of Rz gM) when experiments were repeated with different batches of were determined. The arrowhead indicates the ribozyme concentration riboyzme over a period of 2 years. (5 used to determine the effects of pH on cleavage rate. gM) (B) S (0.13 was incubated with 5 ,uM Rz at the indicated pH. gM) Reactions were carried out as described in Materials and methods. Discussion 'optimized' reaction conditions containing 50 mM Tris, We have modified the natural intramolecular cis-cleavage pH 8.0, 2 mM spermidine, 25 mM KCl and incubated at reaction of VS RNA by constructing a ribozyme containing 30°C (Figure SF). Under these conditions 10 mM MgCl2 144 nucleotides of VS RNA that is capable of an inter- allowed the same rate of cleavage as a reaction containing molecular trans-cleavage reaction. This ribozyme acts as 70 mM MgCl2 under suboptimal conditions (compare a true enzyme in cleaving a 32 nucleotide substrate RNA. Figure SF and B). Thus, the combined effects of tempera- In the presence of excess substrate, the initial rate of ture, pH and cations other than magnesium enhanced cleavage was proportional to ribozyme concentration and cleavage substantially compared with the arbitrary condi- a single ribozyme molecule could cleave multiple substrate tions used for the reactions shown in Figure 1. However, molecules. The was specific in cleaving a single ribozyme no reaction was observed in the absence of MgCl2, the same in phosphodiester bond, one as cleaved the indicating that neither spermidine nor KCl can replace natural cis-cleavage reaction. The trans-cleavage reaction magnesium in cleavage. exhibits Michaelis-Menten with 0.13 iM kinetics, Km and 0.7/min. Fedor and Uhlenbeck have kcat (1990) Effects of pH under single turnover conditions noted that kcat values in the range of 1/min and Km values The trans-cleavage reaction rate showed only a small pH in the nanomolar range are characteristic of many diverse dependence at equimolar concentrations of ribozyme and ribozymes. However, these measurements of kinetic para- substrate (Figure SE). However, these experiments were meters, including ours, used ribozymes removed from their performed at subsaturating concentrations of MgCl2 and natural sequence contexts and intracellular environment. they were probably not under single turnover conditions. Also, the rate-limiting steps may be different in the in in Consequently, it was possible that some step the reaction reactions of the different ribozymes. The similarities Km unless there is other than the actual cleavage step itself may have been and kcat may therefore be coincidental, the the effect of feature of that rate-limiting step, thereby masking some interesting but unrecognized biology increased hydroxide ion concentration. To investigate this results in selection for kinetic in steady-state parameters turnover conditions were established this possibility, single range. under reaction conditions The shortest region of VS RNA that func- empirically optimized by contiguous the initial rates of of 0.13 iM tions as a substrate in the trans reaction described here measuring trans-cleavage concentrations of contains a nucleotide of the site substrate by increasing ribozyme. Figure single upstream cleavage characteriza- 6A shows that the initial rate of increased with and 19 nucleotides downstream. Our cleavage previous showed that a concentration to -2.5 iM and tion of the reaction also only ribozyme up subsequently cis-cleavage of the site leveled that the reaction was nucleotide is off, suggesting approaching single required upstream cleavage in VS is similar to HDV turnover conditions. The rate as a function et this respect single cleavage (Guo al., 1993); which also a of concentration of MgCl2 was re-investigated using ribozymes, require only single upstream be the for cis- or and 0.13 iM S and Rz and found to nucleotide trans-cleavage (Perrotta Been, 5 ,uM essentially not a concentra- Based on minimum free same as in SF 1990, 1992, 1993). energy predic- shape Figure (data shown); and the aberrant tion of 25 mM MgCl2 was chosen to ensure that magnesium tions, electrophoretic mobility pattern we reactions 0.13 of to was not limiting. Trans-cleavage using ,uM accessibility single-strand-specific nucleases, substrate RNA consists of a of showed a minor conclude that the VS mostly S and Rz over a 5 ,uM range pH only structure flanked three nucleotides in reaction rate stable by enhancement (Figure 6B). stem-loop which be involved in 5' and 3' some of on the ends, may this structure reaction kinetics non-Watson-Crick (Figure 4B). Although Steady-state under the conditions of of it was structure to if Rz is appears predominate To determine capable multiple turnover, do not know that it is for molar excess of S the we necessary with an -20-fold reaction, incubated (Figure remains in this structure nor that the RNA molecule cleaved a If each cleavage, 7A). ribozyme only single of the effect the reaction. of 5% of S could be cleaved. In a maximum throughout Indeed, analysis substrate, 373 H.C.T.Guo and R.A.Collins B. A. C. 0. 1.2 0.16 1.0 __ Vmax _ .-f a1) 0.12 .' 0-0 a) E 8 0.6 6, -~0.' -.. O / E o 0'4 0.08 o.~ to. o-00 0.0.4- CIO > 0.04 0.13pM Km= 0 > 0.2 ) l o.. .. . . 0.0 0 0.02 0.04 0.06 0.08 0.2 0.4 0.6 0.8 time (min) [Rz] M [S] PM Fig. 7. Steady-state kinetics. Unless otherwise reactions were stated, carried out as described in Materials and methods at in mM 30°C Tris-HCl, pH 8.0, 5 mM 2 mM MgCl2, spermidine, 25 mM Rz is of KCl). (A) capable turnover. An -20-fold molar excess S multiple (-0.8 RM) was incubated with Rz (0.04 ,uM) and the fraction of substrate cleaved was determined at different times. Rate of is (B) cleavage proportional to ribozyme concentration in the presence of excess substrate. S was (0.75 incubated with 0.04 or 0.08 jM Rz ,uM) 0.01, and the initial rates 0.02, of cleavage were measured. (V0) (C) Determination of and Initial rates of Km of various concentrations of S Vmax. cleavage 0.01 jiM Rz by were determined. Substrate concentrations were 750 and 1000 nM. 2, 10, 50, 125, 250, was calculated as kcat divided V1max (0.14 pmol/min/20 pl) by [Rz] (0.2 pmol/20 ,ul). of base substitution mutations on In addition to in base cis-cleavage this interactions are pairing, tertiary stem-loop region (J.Olive and known or R.A.Collins, to contribute to unpublished suspected substrate binding by results) has revealed a of several complex pattern effects on et et ribozymes (Pyle al., 1992; Dib-Haij al., rate. cleavage Disruption of some base in the In pairs interactions alone stem 1993). fact, tertiary are sufficient to certain by single base substitutions has little or no allow weak > but effect very 0.1 of (Km mM) specific binding on cis-cleavage. However, at some the the of a positions P1 I intron to its identity stem-loop Group core catalytic of one of the bases in a is and particular pair (Doudna RNase P also critical; Szostak, 1989). recognizes even when the compensating substitution is made in substrates that the contain substantial secondary structure and complementary position to restore the have limited helix, cleavage is for Watson-Crick very potential with pairing not restored. We have just begun to the effect the study of et Westhof ribozyme (Smith al., 1992; and Altman, these mutations on trans-cleavage. Our current information We are more direct 1994). currently undertaking measure- suggests that specific bases at specific positions are more ments of the of and mutant binding wild-type substrates important than the to simply presence of a the idea stem-loop investigate that the high apparent affinity of structure. the VS for its ribozyme inferred from its substrate, low The stem-loop structure of the VS substrate RNA is due to interactions. Km (0.13 multiple gM), leaves no long regions available for The Watson-Crick pairing of the temperature optimum trans-cleavage reaction with the ribozyme. Our model for the is working structure lower than for the substantially cis-cleavage reaction of the ribozyme, inferred from the recently determined versus and (30 off much more -45°C) activity drops secondary structure of the minimal at cis-cleaving region of sharply higher temperatures (cf. Collins and Olive, VS RNA (see Figure 4C), predicts that the The ribozyme also retention of at 1993). activity higher temperatures in has no long single-stranded regions. This is in contrast the to cis-cleavage reaction indicates that the active site of most trans-acting ribozymes derived from the does hammerhead, ribozyme not to denature begin until at least hairpin, HDV and Group I intron RNAs, which have been 45°C. The lower optimum temperature of the trans- to designed interact with single-stranded regions of their reaction cleavage reflect decreased may binding of the substrates via formation of one or two intermolecular substrate at higher temperatures. helices flanking the site to be cleaved. In retrospect, it is We noted in our previous characterization of the VS fortunate that we constructed the trans RNA reaction in an reaction cis-cleavage that the cleavage rate was empirical fashion, rather than designing it based on know- unaffected essentially by pH (Collins and Olive, 1993). ledge of the secondary structure of VS RNA. Given the Consistent with this observation, the trans-cleavage precedents from most other ribozymes, it is unlikely that reaction described here also showed little, if any, pH we would have chosen to divide the VS sequence where even dependence, when examined under single turnover we did and expect the two RNAs to interact well. Indeed, conditions. These observations differ from results examin- after we determined the secondary structure of VS RNA, the ing rate of the chemical cleavage step of hammerhead we attempted to design a trans-reaction analogous to other ribozymes (Dahm and Uhlenbeck, 1993), RNase ribozymes, by dividing the VS sequence within loop I, (Guerrier-Takada et al., 1986; Smith and Pace, 1993; however, this combination of RNAs showed no activity Beebe and Fierke; 1994) and Tetrahymena Group I intron (A.Kawamura and R.A.Collins, unpublished results). This et (Herschlag al., 1993; Herschlag and Khosla, 1994). For could be due to inactive RNA conformations in the these the rate ribozymes, of the cleavage step was found ribozyme and/or substrate, or it may indicate that to increase with increasing pH. Failure to observe pH sequences in loop I are important for formation of the in dependence VS RNA could mean that OH- is not active structure. involved in the cleavage reaction, that the reaction 374 Trans-cleavage by a VS RNA ribozyme NaCl, 2 mM proceeds via a novel mechanism or, more likely, that the 300 U RNAguard spermidine-(HCl)3], (Pharmacia), 150- 200 U RNA T7 polymerase (Bethesda Research Laboratories) for 2 h VS trans reaction is not limited by the rate of the chemical at 37°C. Radioactive transcripts were prepared as above except an cleavage step under these conditions, but rather by some additional 30 jiCi [cx-32P]GTP (or, for specific experiments, ATP or step that precedes actual cleavage. UTP) was added. Samples were subsequently treated with DNase I (5 U/ jg DNA template; One interesting candidate for such a rate-limiting step Pharmacia) for 15 min, then EDTA was added to 10 mM. RNAs were extracted with would be a change in the substrate and/ phenol:chloroform:isoamyl alcohol, conformational chloroform:isoamyl alcohol (CIA) and in the ethanol-precipitated or nbozyme following binding. At saturating ribozyme presence of 0.3 M sodium acetate, pH 5.2. concentration, the pseudo-first-order rate constant for Precipitated RNAs were dissolved in water and two volumes of trans-cleavage of S (-0.6/min) is -10-fold higher than sequencing dye (95% formamide, 0.5X TBE, 0.1% xylene 0.1% cyanol, bromphenol blue), heated at the rate of cis-cleavage of RNA under similar 75°C for 3 min and fractionated GIl by electrophoresis on denaturing polyacrylamide gels (40:1 acrylamide:bis- conditions (determined from plots of fraction of uncleaved acrylamide) of appropriate 1 concentration containing 8.3 M urea and x RNA versus time of the data used for Figure 6 and Collins TBE (135 mM Tris, 45 mM boric mM RNAs were acid, 2.5 EDTA). and Olive, 1993). Since we envision that the trans- visualized either by autoradiography or UV Bands of interest shadowing. were excised, cleavage reaction recreates essentially the same RNA eluted overnight at 4°C in water and filtered to remove residual polyacrylamide. RNAs were precipitated with ethanol in the conformation as in the cis-cleavage reaction, the higher presence of 0.3 M sodium acetate and dissolved in water. Concentrations rate suggests that the cleavable conformation may be more were determined 1 spectrophotometrically, assuming corresponds OD260 easily attained when S (stem-loop I; Figure 4C) is not to an RNA concentration of 40 ,ug/ml. constrained by covalent attachment to the nbozyme core. End-labeling of RNAs In support of this idea, we have also found that the rate RNAs were labeled at 5' T4 kinase and termini using polynucleotide of cis-cleavage of GIl RNA can be increased several fold or at T4 RNA and End- 3' termini using ligase [y-32P]ATP 5'-[32P]pCp. by increasing the distance between stem-loop I and the labeled RNAs were on and fractionated denaturing polyacrylamide gels ribozyme core (A.Andersen and R.A.Collins, unpublished detected by autoradiography. In order to remove to 5' some results). These observations are consistent with the idea 5'-triphosphates prior end-labeling, RNAs were treated with U calf intestinal alkaline phosphatase of at least one conformational change involving the (Boehringer Mannheim) in a 10 p1 reaction 50mM containing Tris-HCI, substrate stem-loop occurring during the reaction. pH 8.0, 0.1 mM EDTA at 55°C for 30 min. Reactions were terminated The observation that the VS can recognize a ribozyme by extraction with phenol:CIA and CIA. substrate that contains a stable secondary structure may RNA sequencing be useful from the perspective of ribozyme engineering. End-labeled RNAs were partially digested with RNase TI or U2 Among the limitations to modifying hammerhead, hairpin essentially as described by Donis-Keller et al. in (1977) denaturing or Group I intron riboyzmes to cleave non-native target buffer (7 1 M urea, 50 mM citric acid, mM 0.05% EDTA, xylene cyanol, RNAs is the requirement that the target site be in a single- 150 yeast 5 jig/ml tRNA) and adjusted with NaOH to for TI pH digests and pH 3.5 for in ice. To stranded region to allow recognition via base pairing with U2. Reactions were terminated by chilling prepare partial RNA and 0.5 alkaline hydrolysis products, labeled the ribozyme. Because the cleavage site for the VS RIg/l yeast carrier tRNA for in 0.15 M were heated to 95°C 5-10 min NH40H, ribozyme is adjacent to a stable secondary structure, the lyophilized and dissolved in buffer. RNAs were denaturing separated by VS ribozyme may have unique properties that can be electrophoresis on 20% denaturing polyacrylamide gels. adapted to cleaving certain RNAs that are not accessible Structure probing to the action of other ribozymes. We are currently Limited partial digestion of 5' end-labeled S was under native performed investigating the substrate recognition requirements, conditions essentially as described Labeled RNA by Knapp (1989). (10- including whether substrate secondary structure is 100 ng) jl mM was dissolved in 27 of 50 mM 25 Tris-HCI, pH 8.0, required, or simply tolerated, by the ribozyme. tRNA. KCI, 10 mM MgC12, 2 mM 2.5 spermidine-(HCl)3, jg/jl yeast the reactions were RNase TI (4.5 U) or T2 (0.225 was added and U) incubated removed at 15 and at 30°C. Samples (3 jl) were 0.5, 2, 3, 8, and mixed Materials and methods 30 min (RNase T1) or 2, 3, 15 30 min 5, 8, (RNase T2), with an M 10% 0.05% equal volume of stop solution (9 urea, glycerol, DNA templates and synthesis of RNAs in ice. Products were to xylene cyanol) and frozen dry subjected 20% followed Fragments of VS DNA were cloned into vectors pTZ18R or 19R electrophoresis on denaturing polyacrylamide gels by (Pharmacia). Clone Gl (see Guo et al., 1993) contains VS nucleotides autoradiography. 617-881, numbered as in Saville and Collins (1990); the cleavage site is between G620 and A621. Substrate RNAs were transcribed (see Analysis of 5' and 3' termini nuclease below) from or its derivatives which had been linearized at the Identification of 5' and 3' terminal nucleotides P1 GlI by digestion labeled AvaI site (nucleotide 639) or the SspI site (nucleotide 783) to make and thin of two-dimensional layer chromatography appropriately RNAs designated GlIl/AvaI and GIl/SspI respectively. These RNAs RNAs was carried out as described previously 1967; (Shugar, Silberklang begin with nine vector nucleotides (5'-gggaaagcu) followed by VS et al., 1979; Saville and Collins, 1990). sequence. A site-directed mutant of G 11, clone 621 U, which contains a single A-*U substitution immediately following the self-cleavage site, Trans-cleavage reactions AvaI was carried out was also used. Trans-cleavage of S by the ribozyme (Rz) following X reaction S and Rz in the 1 Clone A-3 contains VS sequences downstream of the AvaI site pre-incubation of gel-purified appropriate addition of to Reactions were initiated (nucleotides. 640-881) in a derivative of pTZ19R that lacks the XbaI solution for 2 min. by ribozyme jl. In a 10 of in a final volume of 20 reaction, and SphI sites in the multiple cloning site (constructed for reasons substrate typical aliquots terminated addition of 13.5 jl unrelated to the project described here). Transcripts of clone A-3 digested 1.5 jl were removed at specified times, by 7 mM 0.07% with SspI (VS nucleotide 783) begin with 9 vector nucleotides (5'- of stop mix (70% EDTA, 0.4X TBE, formamide, xylene and stored on ice. were gggaaagcu) followed by 144 nucleotides of VS RNA; this RNA is cyanol, 0.07% bromphenol blue) Samples 20% on designated the AvaI ribozyme, or Rz. fractionated by electrophoresis gels. denaturing polyacrylamide and on The RNAs were prepared by in vitro T7 RNA polymerase transcription effects of temperature, pH, MgCl2 spermidine-(HCl)3 were the reaction from linearized plasmid DNAs. Transcription reactions (usually 300 jl) trans-cleavage incubating equimolar analyzed by jiM in solutions as described in concentrations of Rz and S contained icg appropriately linearized template, mM NTP (0.05 each) 10-20 1 each the effects of and For no (Pharmacia), 5 mM dithiothreitol, Ix T7 polymerase buffer [Bethesda the reason, NaCl KCl good figure legends. jM S. A final of the jM Rz and 0.125 at 0.05 Research Laboratories; 40 mM Tris-HCI, pH 8.0, 8mM MgCi2, 25 mM were examined study 375 H.C.T.Guo and R.A.Collins effects of MgCl2 under otherwise conditions was and 'optimized' Perrotta,A.T. Been,M.D. Nucleic Acids 3959-3965. performed (1993) Res., 21, at 30°C, 50 mM 2 mM 25 mM and Tris-HCI, pH 8.0, spermidine, KCI. Cech,T.R. 123-128. Pyle,A.M., Murphy,F.L. (1992) Nature, 358, Experiments to establish turnover conditions PhD single (Figure 7A) Saville,B.J. thesis, of Toronto. were (1991) University performed at in 50 mM 30°C 25 mM 25 and Tris-HCI, pH 7.1, mM Saville,B.J. Collins,R.A. Cell, 61, 685-696. (1990) MgC92, KCI, 2 mM spermidine. Analyses of the effect of under pH single Sharmeen,L., Kuo,M.Y.P., and J. Dinter-Gottleib,G. Taylor,J. (1988) turnover conditions (Figure 6B) were performed as 2674-2679. above, except that Virol., 62, the concentrations of Rz and S were 5 IM and 0.13 jM Methods respectively. Shugar,D. (1967) Enzymol., 12A, 131-137. Tris-HCI (50 was mM) used for pHs 7.1-8.9; 16.5 mM PIPES/44 and mM Silberklang,M., Gillum,A.M. Methods RajBhandary,U.L. (1979) Tris (Smith and Pace, was used for 1993) pH 6. 58-109. 19, Enzymol., Amounts of substrate and were products quantitated a and using Phosphor- Smith,D. Pace,N.R. (1993) Biochemistry, 5273-5281. 32, Imager and ImageQuant version 3.0 software (Molecular and Dynamics, Smith,D., Burgin,A.B., Haas,E.S Pace,N.R. (1992) J. Biol. Chem., Sunnyvale, CA). Estimates of initial rates cleavage were derived from 2429-2436. 267, plots of fraction of substrate cleaved versus time using Grafit software Symons,R.H. Annu. Rev. (1992) Biochem., 61, 641-671. (Erithacus Software Ltd, Staines, UK). Up to 90% of the substrate could Szostak,J. 83-86. (1986) Nature, 322, be cleaved in 60 min at concentration approximately equimolar of Uhlenbeck,O.C. (1987) Nature, 328, 596-600. ribozyme, with the curve indicating the presence of -10% and unreactive Westhof,E. Altman,S. Proc. Natl Acad. Sci. (1994) USA, 91, starting material. Curves were not adjusted to 100% and 5133-5137. completion the nature of the unreactive substrate has not been characterized further. Wu,H.-N, Wang,Y.-J., and Hung,C.-F., Lee,H.-J. Lai,M.M.C. (1992) J. 233-245. MoL Biol., 223, and Zaug,A.J. Cech,T.R. (1986) Science, 231, 470475. Ackcnowledaements Zuker,M. (1989) 48-52. Science, 244, We thank Murray Schnare for advice on ribonuclease sequencing, Anne Received on August 9, 1994; revised on October 1994 24, Kawamura for with help some of the single turnover experiments and Joan Olive for 3B. We Figure also thank Olke Debbie Uhlenbeck, Field, Diane De Abreu, Elisabeth Tillier and Jim Hogan for helpful discussions and Tom Cech for comments on the This work was manuscript. supported by an Operating Grant from the Medical Research Council of Canada. H.C.T.G. was a recipient of a NSERC Postgraduate Scholarship. References and Beebe,J.A Fierke,C.A. (1994) Biochemistry, 33, 10294-10304. Branch,A.D. and Robertson,H.D. (1991) Proc. Natl Acad. Sci. USA, 88, 10163-10167. Buzayan,J.M., Gerlach,W.L. and Bruening,G. (1986) Nature, 323, 349-353. and Castanotto,D., Rossi,J.J. Deshler,J.O. Crit. Rev. (1992) Eukaryotic Gene 331-357. Expression, 2, Cech,T.R. (1990) Annu. Rev. Biochem., 59, 543-568. Collins,R.A. and Olive,J. (1993) Biochemistry, 32, 2795-2799. Collins,R.A. and Saville,B.J (1990) Nature, 345, 177-179. Dahm, S.C. and Uhlenbeck,O.C. (1993) Biochemistry, 32, 13040-13045. Dib-Haij,S.D., Boulanger,S.C., Hebbar,S.K., Peebles,C.L., Franzen,J.S. and Perlman,P.S. (1993) Nucleic Acids Res., 21, 1797-1804. Donis-Keller,H., Maxam,A.M. and Gilbert,W. (1977) Nucleic Acids Res., 4, 2527-2538. Doudna,J.A. and Szostak,J.W. (1989) Nature, 339, 519-522. Fedor,M.J. and Uhlenbeck,O.C. (1990) Proc. Natl Acad. Sci. USA, 87, 168-1672. Feldstein,P.A. and Bruening,G. (1993) Nucleic Acids Res., 21, 1991- Feldstein,P.A., Buzayan,J.M. and Breuning,G. (1989) Gene, 82, 53-61. Feldstein,P.A., Buzayan,J.M., van Tol,H., de Bear,J., Gough,G.R., and Gilham,P.T. Bruening,G. Proc. Natl (1990) Acad. Sci. USA, 87, 2623-2627. Forster,A.C. and Symons,R.H. (1987) Cell, 50, 9-16. Guerrier-Takada,C. and Altman,S. (1993) Biochemistry, 32, 7152-7161. Guerrier-Takada,C., Gardiner,K., Marsh,T., Pace,N. and Altman,S. (1983) Cell, 35, 849-857. Guerrier-Takada,C., Haydock,K., Allen,L. and Altman,S. (1986) Biochemistry, 25, 1509-1515. Guo,H.C.T. (1992) MSc Thesis, University of Toronto. Guo,H.C.T., De Abreu,D., Tillier,E.M., Saville,B.J., Olive,J.E. and Collins,R.A. (1993) J. MoL Biol., 232, 351-361. Hampel,A., Tritz,R., Hicks,M. and Cruz,P. (1990) Nucleic Acids Res., 18, 299-304. Herschlag,D. and Khosla,M. (1994) Biochemistry, 33, 5291-5297. Herschlag,D., Eckstein,F. and Cech,T.R. (1993) Biochemistry, 32, 8312-8321. Knapp,G. (1989) Methods Enzymol., 180, 192-212. Kuo,M.Y-P., Sharmeen,L., Dinter-Gottlieb,G. and Taylor,J. (1988) J. Virol., 62, 4439-4444. Michel,F., Umesono,K. and Ozeki,H. (1989) Gene, 82, 5-30. Perrotta,A.T. and Been,M.D. (1990) Nucleic Acids Res., 18, 6821-6827. Perrotta,A.T. and Been,M.D. (1991) Nature, 350, 434-436. Perrotta,A.T. and Been,M.D. (1992) Biochemistry, 31, 16-21. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Efficient trans‐cleavage of a stem‐loop RNA substrate by a ribozyme derived from neurospora VS RNA.

Guo, H.C.; Collins, R.A.
The EMBO Journal , Volume 14 (2) – Jan 1, 1995
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Springer Journals
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Copyright © European Molecular Biology Organization 1995
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0261-4189
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1460-2075
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10.1002/j.1460-2075.1995.tb07011.x
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Abstract

EMBO Journal vol.14 no.2 1995 The pp.368-376, of a stem RNA Efficient trans-cleavage -loop a derived from substrate by ribozyme Neurospora VS RNA et al., 1990) and HDV (Branch and Robertson, 1991; C.T.Guo and Richard A.Collins1 Hans Perrotta and Been, 1992; Wu et al., 1992) ribozymes Canadian Instutute for Advanced Research Program in Evolutionary have been facilitated by altering these RNAs to perform Biology, Department of Molecular and Medical Genetics, University of intermolecular trans-cleavage reactions. In a trans- Canada M5S IA8 Toronto, Toronto, Ontario, cleavage reaction one RNA, the substrate, contains the author 'Corresponding site to be cleaved; a separate RNA, the ribozyme, provides Communicated by T.R.Cech the to the One sequences required catalyze cleavage. has been naturally occurring trans-acting ribozyme a 144 We have constructed ribozyme containing the RNA of RNase which discovered, component P, VS RNA that can nucleotides of Neurospora catalyze in trans cleaves pre-tRNA precursors (Guerrier-Takada of a RNA in a true the cleavage separate enzymatic reactions of most et al., 1983). Trans-cleavage ribozymes manner 0.13 0.7/min). have been such that of the substrate (Km ,uM, kcat Comparison designed binding of the rates of cis- and as well as the trans-cleavage, occurs via formation of Watson-Crick base multiple lack of effect of pH on the rate of cleavage, suggest with the Non-Watson-Crick and pairs ribozyme. tertiary that a rate-limiting step, possibly conformational interactions are also involved in substrate and binding The minimum con- change, occurs prior to cleavage. for et may be essential proper binding (Pyle al., 1992; for con- tiguous substrate sequence required cleavage et Guerrier-Takada Smith et al., 1992; Dib-Haij al., 1993; and 19 nucleotides sists of one nucleotide upstream and Altman, 1993). the site. Unlike most other downstream of cleavage and I it With hammerhead, hairpin Group ribozymes which interact with ribozymes long single-stranded nucleotides in the has been found that very few specific the minimal substrate for regions of their substrates, that substrate are required for trans-cleavage, provided consists of a stable the VS ribozyme mostly stem-loop, the adjacent region(s) are complementary to the binding which would to its appear preclude recognition simply the site on the ribozyme. This property has allowed via extensive Watson-Crick base pairing. engineering of ribozymes that can cleave sequences other Key words: cleavage/Neurospora/plasmid/RNA/ribozyme than those recognized by the naturally occurring ribozyme. Some also function in vivo in non- engineered ribozymes native host cells, which has raised the possibility of their use as therapeutic agents in dominant inherited disorders Introduction and against retroviruses and RNA viruses (reviewed by Castanotto et al., 1992). A small number of RNAs isolated from a of variety We are the VS RNA which is found in studying catalytic natural sources have been found to a possess self-cleavage the mitochondria of certain natural isolates of Neurospora that is involved in multimeric tran- activity processing and VS the (Collins Saville, 1990). performs same type scripts into monomers, apparently as part of the replication of RNA as cleavage hammerhead, hairpin and HDV cycle. Several different RNA sequences and secondary ribozymes, leaving products with 2',3'-cyclic phosphate structures appear to be capable of such activity. These and 5'-OH termini and include: in (Saville Collins, 1990), but it the hammerhead, found several plant viral is different in sequence, secondary structure, choice of satellite a RNAs, viroid RNA and the transcript of a cleavage site and functional properties from these other nuclear satellite DNA of a newt (Forster and Symons, ribozymes (Collins and Olive, 1993; Guo et al., 1993; 1987; reviewed by Symons, 1992); the hairpin (or paper T.L.Beattie, J.E.Olive and R.A.Collins, submitted). An clip) in the minus strand of the satellite of tobacco ringspot understanding of the structure and function of VS RNA virus and related viruses (Buzayan et al., 1986; Feldstein may therefore provide unique information about the ways et al., 1990; Hampel et al., 1990; Feldstein and Breuning, 1993); the genomic and antigenomic RNAs of hepatitis that RNAs can catalyze reactions and about the nature of delta virus (HDV; Kuo et al., 1988; Sharmeen et al., 1988; RNA active sites. Perrotta and Been, 1991, 1993); and VS RNA in the We report here that the catalytic region of VS RNA mitochondria of certain Neurospora isolates (Saville and can cleave a separate substrate RNA at a specific target Collins, 1990). site. The minimal substrate forms a stable hairpin with In their natural contexts the ribozymes mentioned above, few and only short single-stranded regions potentially as well as others such as Group I (Cech, 1990) and Group available for recognition by the ribozyme via Watson- II introns (Michel et al., 1989), perform intramolecular Crick pairing. Nonetheless, the low Km of the VS cis-cleavage and, in some cases, ligation reactions. trans-cleavage reaction (0.13 ,uM) suggests that the VS Structure-function studies of Group I introns (Szostak, ribozyme binds its substrate quite efficiently, implying 1986; Zaug et al., 1986) and later hammerhead that substrate recognition involves multiple, including (Uhlenbeck, 1987), hairpin (Feldstein et al., 1990; Hampel tertiary, interactions. 38 Oxford University Press Trans-cleavage by a VS RNA ribozyme Results of Gl 1/Ava substrate (S) A. sequence VS RNA can cleave a separate RNA substrate in gggaaagcuUGCGAAGGGCGUCGUCGCCCCG A trans 5 A 3, The trans reaction described below was constructed before cleavage site we had determined the secondary structure of VS RNA. In the absence of structure-based predictions about where B. to divide the normally cis-cleaving RNA to establish a trans-cleaving system, we used several restriction frag- S (32 nt) 3' ments of VS DNA cloned in a T7 promoter vector to + Ribozyme (Rz) construct pairs of non-overlapping regions of VS RNA. One member of each pair, the substrate (S; see Materials PI (13 nt) and methods), contained the expected cleavage site, fol- P2 (19 nt) lowing nucleotide G620 (numbered as in Saville and Collins, 1990); the other, the enzyme or ribozyme (Rz; RNA s S + Rz see Materials and methods), contained the remainder of the VS sequence, terminating at the SspI site at nucleotide time (min) 0 1 2 4 6 101530 60 783. In preliminary experiments (Saville, 1991; Guo, 1992) these transcripts were mixed at an 1:1 ratio and &&A A incubated under conditions known to support cis-cleavage (Collins and Olive, 1993). Most combinations showed little or no cleavage, however, almost complete cleavage of a 32 nucleotide substrate RNA that terminates at the AvaI site (nucleotide 639) was observed during a 1 h incubation with a ribozyme that begins at the AvaI site P2 ~ u4 _ and ends at the SspI site (nucleotide 783); no cleavage was observed in the absence of ribozyme (Figure 1). The electrophoretic mobilities of the two cleavage products, P1 ~ o were approximately those expected for cleavage P1 and P2, (confirmed below), which is the site after nucleotide 620 cis-cleavage of VS RNA. We chose to of intramolecular Fig. 1. The trans-cleavage reaction. (A) The nucleotide sequence of the GI l/AvaI substrate RNA (S) transcribed from clone GIl which this trans-cleavage reaction in further detail. examine with AvaI. VS nucleotides 617-639 had been linearized by digestion and Collins, 1990) are indicated in upper case; (numbered as in Saville are in lower case. (B) Time course of trans- vector nucleotides occurs at the same site as cis- Trans-cleavage (Rz). 32P-Labeled S and unlabeled cleavage of S by the AvaI ribozyme cleavage were incubated in 50 mM Tris-HCl, pH 7.5, Rz (0.05 jiM each) determine the precise site of cleavage, S, P1 and P2 To at 37°C. Aliquots were removed at the times indicated 20 mM MgC12 at their 5' ends and sequenced by partial were labeled electrophoresis and autoradiography (see Materials and analyzed by for details). The left lane contains a control incubation in using RNases TI or U2 (Figure 2). and methods enzymatic digestion of Rz. P1 and P2 are the cleavage products of S. the absence of a mutant substrate containing a Cleavage products substitution 3' of the cleavage site (A621U) single base between nucleotides 620 and 621, as in the cis- to resolve possible ambiguities occurred were also characterized reaction. to anomalous migration of some bands (see below). cleavage due and P1 are identical in sequence from the 5' Because S Minimal length of the substrate RNA the site, all RNase sequencing bands co- end to cleavage downstream To determine the minimal sequence required as (Figure 2A-C). Full-length P1 co- migrated, expected we used essentially the approach of the cleavage site, with the 13 nucleotide RNase TI fragment of S migrated and (1987) and used for described by Forster Symons that terminates at G620, which is the site of intramolecular et al. Perotta and Been, other riboyzmes (Feldstein 1989; in VS RNA. Also, the 3' end of P1 was cis-cleavage RNA was partially 1992). 5'-End-labeled G11/SspI to be 2',3'-cyclic phosphate (Figure 2F), found guanosine treatment at high pH, then incubated with location and chemical pathway of hydrolyzed by that both the indicating 3A). Incubation in the same as in the reaction. or without the ribozyme (Figure are the cis-cleavage trans-cleavage confirmed our 2) previous of a at absence of ribozyme (lane As from the finding cyclic phosphate expected Gi RNA and deletion was found at the that full-length 1/SspI end of a 5' group finding the 3' P1, hydroxyl < 10 nucleotides at the 3' end can cis- evidenced its derivatives lacking 5' end of as by end-labeling by P2, Incubation with the ribozyme et 1993). T4 kinase without prior cleave (Guo al., and polynucleotide [,y-32P]ATP or at least decrease in resulted in the disappearance, Alkaline ladders of 5' treatment. hydrolysis phosphatase to RNAs terminating at of bands corresponding P2 contained 18 of the expected 19 intensity, end-labeled only A few RNAs were not or nucleotide 639 longer (lane 4). This is the result of a artefact involving bands. compression these under conditions, indicating cleaved to completion of a stable stem-loop structure in the the formation very substrates. The minimal are poorer that they relatively this will be described in detail below. longer RNAs; terminates at residue which by substrate 639, 5' terminal nucleotides of P2 derived length the Nonetheless, to the RNA used in precisely S and the A621U mutant were A and U coincidence corresponds of from cleavage run-off was transcription which by 2G and that cleavage Figure 1, synthesized H), confirming respectively (Figure 369 and R.A.Collins H.C.T.Guo E. D. A. B. C. P2 G 1 P2 621U Gll S G1 Pl 621U P1 1 23A45 1 23 A45 12 3A45 12345 12345 G638 G638 G630 G633 -G62<8 G618 G625 G624 a * a , G623 9 eM *. F. G. H. Ribonuclease TI and U2 of 5' end-labeled S and of the sequencing cleavage products P1 Fig. 2. Characterization trans-cleavage products. (A-E) or incubated in TI or U2 buffer without RNases lanes clone GIl and mutant 621U. Lanes 1-3 are controls respectively); A, and P2 from (untreated RNase lanes RNase U2. Nucleotide are indicated to the left of the VS nucleotides alkaline lanes TI; 5, sequences figure. partial hydrolysis ladder; 4, in the site of is indicated the arrowhead. Thin of: the are in vector nucleotides lower cleavage by (F-H) layer chromatography (F) upper case, case; of 5' GIl the 5' nucleotide of 5' 621U P2. 3' nucleotide of GIl P1; (G) the 5' nucleotide phosphorylated P2; (H) phosphorylated of the 1 linearized at the AvaI site. Thus 19 characterization trans-cleavage products (Figures of a template only that were consistent nucleotides downstream of the cleavage site are required and 2) we noted several observations P2 than expected for the AvaI with such a structure. migrated faster trans-cleavage by ribozyme. RNA relative to size markers for a 19 nucleotide RNA, sug- A experiment using 3' end-labeled showed parallel of the site that only a single nucleotide upstream cleavage gesting that it contained a structure that was not fully for Taken together denatured even in a gel containing 8.3 M urea (Figure 1 is required trans-cleavage (Figure 3B). with the results from 5' end-labeled RNA 3A), and data not shown). Certain guanosine (623-625, 627 (Figure these data show that the minimum contiguous region of and and adenosine and 622) residues in S and 633) (621 the native RNA required for trans-cleavage consists of P2 were cleaved weakly or not at all by RNases TI and/ one nucleotide of the and 19 upstream cleavage site or U2, even though sequencing reactions were performed nucleotides downstream. under putatively denaturing conditions of 50°C, 1 mM EDTA and 7 M urea (Figure 2A-E). Only 18 of the The minimal substrate RNA consists mostly of a expected 19 bands were observed in the 5' end-labeled stable hairpin loop alkaline hydrolysis products of P2 (in the gel shown partial RNA structure prediction using the MFOLD program of in Figure 2D and E, the first nucleotide, A621, has been Zuker and collaborators (Zuker, 1989; data not shown) run off the gel, so only 17 bands are shown). Also, the suggests that the most thermodynamically reasonable spacing and intensity of bands at C634-C636 was uneven structure of the substrate RNA would be the hairpin- (Figure 2D and E), typical of compression artefacts during containing structure drawn in Figure 4B. During the caused by a secondary structure in the electrophoresis 370 a Trans-cleavage by VS RNA ribozyme A. cleavage B. cleavage site B.site G620 U783 G620 A639 52 p _* _ 5t- 3T 35$cp partial alkaline partial partial partial hydrolysis parl partial alkalineT Ti hydrolysi's digestion +Rz digestion -Rz -Rz +Rz markers 60' 0' 60' lane: i 2 3 C D 1' 30' 60' E -O 60' I'll / / 4' \// cleavable RNAs G61 8 G620 " I -w- P2 cleavE,able RNAs 5;; ... :-7 G630 ; +- * * .9 .- F , * _ G640- _ G638- g * '* *4 * .-NW q~ -__ G620- -n P1 G638 * * .23 t? 1 2 3 4 5 6 7 Fig. 3. Minimal substrate requirements for trans-cleavage of VS substrates by the AvaI ribozyme. End-labeled substrate RNAs, diagrammed at the top of each panel, were treated as indicated in the flow charts. (A) 5'-End-labeled GI I/SspI substrate. Substrate (0.3 was incubated with Rz gM) (0.3 in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 at 37°C for 0 or 60 min. (B) gM) 3'-[32P]pCp-End-labeled GI I/AvaI substrate. Substrate (0.05 and Rz (1.5 FM) were incubated as in (A) and samples were withdrawn at the gM) times indicated in lanes 4-7. To facilitate interpretation, relevant guanosines are indicated in the TI marker lane. P1 and P2 mark the position of the detectable (end-labeled) cleavage product in (A) and (B) respectively. longer molecules. The pattern and intensity of bands is although T1, G620 itself was only weakly cleaved. The consistent with the interpretation that the two RNAs in only strong TI cleavages downstream of G620 were at the alkaline ladder that terminate at C635 and C636 G630 in the were loop and G638 in the 3' single-stranded compressed; the RNA terminating at C635 is denatured, region. Guanosines in the stem were resistant to TI like those shorter than it (below it in the gel), while the cleavage. The RNase TI data are not consistent with an RNA ending at C636 extends the putative helix by one alternative structure in which two substrate RNAs dimerize base pair, thereby preventing its denaturation under to form a partially self-complementary duplex. In such these electrophoretic conditions. The duplex, G630 hairpin-containing would be paired and inaccessible to RNase RNA co-migrates with the but shorter, denatured, product TI. RNase T2, which has specificity for single-stranded terminating at C635. regions (Knapp, 1989), cleaved in the loop (U628, C629) results were Complementary obtained from RNase and at some positions upstream of the stem (U617, C619) sequencing of 3' end-labeled S and P2 (data not and weakly at A622. shown), A621, The weak TI and T2 cleavages i.e. the same guanosines and adenosines were resistant at to several positions (620-622) adjacent to the Watson- cleavage by the RNases and the alkaline Crick hydrolysis ladder base pairs in the hairpin stem may indicate that had a compression the two RNAs with 5' some structure involving extends beyond that drawn in Figure 4B. ends at G623 and G624 to C636 (those complementary and C635). Reaction conditions Nuclease under probing native conditions is also con- We have the effects of several variables investigated that sistent with the formation of a stable in S would be expected to affect RNA structure hairpin loop and that have (Figure 4A and data not Guanosines of been shown). found to affect the cleavage rates of other upstream ribozymes. the site at G620 to be We ribozyme cleavage arbitrarily chose an ratio of S and appeared single- equimolar Rz as were to RNase (0.05 stranded, they susceptible iM each) for most initial cleavage by investigations; more 371 H.C.T.Guo and R.A.Collins A. B. A. cu RNase RNase Ti T2 .' 100 oJ S 10 u C 1I 0 20 40 60 0 20 40 60 80 stem 3' mM temp (C) [MgCI2] G633 loop G630 C. D. 100 10Q0 NaCI G627 2E 100' stem 5' KCI E 10 - G625 G624 * S do G623 0 0 .1 .2 .4 2 4 6 8 10 .3 .5 G620 4*0 M [spermidine] mM [monovalent] ip. Ga618 *Q F. 100 'pp.4 100 E 10 \ E,g 7 7.5 8.5 9 0 2 8 4 6 8 10 mM pH [MgCI2] ¶4 5. Effect of Fig. reaction conditions on cleavage of S by Rz. Unless (A-E) otherwise indicated, reactions were carried out as B. Minimal secondary structure of Gl 1/Ava S described in Materials and methods at 37°C in the presence of 50 mM Tris-HCI, 10 mM pH 7.5, MgCl2 and the variables indicated on the 0 L 0 c')v X axes. The effect (F) of [MgCI2] under otherwise 'optimized' CQN NNI D Ut (D CO to to conditions: 50 mM 30°C, Tris-HCI, pH 8.0, 25 mM KC1, 2 mM 5,gggaaagcuUGC spermidine. GMGGGCG cleavage site AGCCCCGC UG3 detailed analyses specifically under either steady-state or co S single turnover conditions are described later. Cleavage rate increased with temperature until an optimum was around reached 30°C and then decreased C. sharply above 40°C (Figure No reaction 5A). was observed . .. ii 11 11 in the absence of a divalent cation and reaction rate increased with increasing MgCl2, reaching a maximum around 100 mM when magnesium was the only cation I present (Figure 5B). To determine whether some of 620 the MgCl2 was as a structural acting simply the counterion, effects of spermidine, NaCl, and KCI were investigated III 1IIl III I II 111111111) 5/ in the presence of a subsaturating concentration of MgCl2 5 3' \ (10 mM). In the presence of 10 mM MgCl2, spermidine at 1 mM or greater enhanced the rate of cleavage nearly 10- fold compared with the same reaction without spermidine 4. Structure of S and Rz (Figure 5C). Low concentrations of Fig. RNAs. (A) Limited RNase TI and T2 KCI (<100 mM) also digestion. 5'-End-labeled S was partially digested with RNase TI or stimulated the reaction rate to up -10-fold. Perhaps T2 at 30°C in native buffer (50 mM Tris-HCI, pH 8.0, 25 mM KCI, surprisingly, NaCl had almost no effect (Figure 5D). These 10 mM MgCl2, 2 mM spermidine) for 0.5-30 min. A guanosine observations are similar to the effects of cations observed marker lane (labeled TI denatured) was Ti prepared by RNase previously on the rate of in cis-cleavage of VS RNA sequencing denaturing buffer (see Materials and methods for (Collins details). Controls 1 and 2 contain RNA incubated in the absence of and The Olive, 1993). stimulation by low concentrations RNase in denaturing buffer or native buffer respectively. OH indicates of some other cations suggests that some MgCl2 was partial alkaline hydrolysis ladder. Nucleotides contributing to the acting as a counterion which could be replaced more stem-loop structure are indicated on the right. (B) Minimal secondary effectively by spermidine or potassium. structure of S. VS are in nucleotides upper case, vector in nucleotides lower case. The box indicates the The rate of minimal length of the substrate reaction showed only a small pH dependence. determined from deletion experiments shown in Figure 3. The nearly 100-fold increase in the hydroxide concentra- (C) Proposed secondary structure of the minimal cis-cleaving region tion between pH 7.1 and 8.9 resulted in only a 2-fold of VS RNA (T.L.Beattie, J.E.Olive and R.A.Collins, submitted). The increase in rate (Figure 5E). The effect of pH specifically box encloses the minimal substrate for the trans reaction (boxed in B). Rz consists of the under single turnover sequences downstream of the conditions is described later. substrate, forming stems II-VI (see Materials and methods). Finally, the effect of MgCl2 was re-assayed under 372 Trans-cleavage by a VS RNA ribozyme contrast, we observed that cleavage continued at a constant A. B. rate until -40% of S was cleaved and then decreased 1.5 slowly as the concentration of available uncleaved S I- decreased. This indicated that Rz behaved like a true l_ A enzyme in that it was capable of multiple rounds of v0 cleavage. Also, as expected of an enzyme, the initial rate 0.6 EL of cleavage was directly proportional to the concentration :0-3 of the ribozyme under conditions of substrate excess z0. (Figure 7B). 0.1II 01- . . . . . . The trans-cleavage reaction exhibits a saturation curve 0 1 2 3 4 5 10 6.0 7.0 8.0 9.0 with respect to substrate concentration that is typical of uM pH [Rz] Michaelis-Menten kinetics (Figure 7C). A Km of 0.13 jM Fig. 6. Effects of pH on the rate of trans-cleavage under single and kcat of 0.7/min were obtained from these data. These turnover conditions. (A) Determining single turnover conditions. Initial values have been observed to vary by a factor of -2 rates of cleavage of S (0.13 by increasing concentrations of Rz gM) when experiments were repeated with different batches of were determined. The arrowhead indicates the ribozyme concentration riboyzme over a period of 2 years. (5 used to determine the effects of pH on cleavage rate. gM) (B) S (0.13 was incubated with 5 ,uM Rz at the indicated pH. gM) Reactions were carried out as described in Materials and methods. Discussion 'optimized' reaction conditions containing 50 mM Tris, We have modified the natural intramolecular cis-cleavage pH 8.0, 2 mM spermidine, 25 mM KCl and incubated at reaction of VS RNA by constructing a ribozyme containing 30°C (Figure SF). Under these conditions 10 mM MgCl2 144 nucleotides of VS RNA that is capable of an inter- allowed the same rate of cleavage as a reaction containing molecular trans-cleavage reaction. This ribozyme acts as 70 mM MgCl2 under suboptimal conditions (compare a true enzyme in cleaving a 32 nucleotide substrate RNA. Figure SF and B). Thus, the combined effects of tempera- In the presence of excess substrate, the initial rate of ture, pH and cations other than magnesium enhanced cleavage was proportional to ribozyme concentration and cleavage substantially compared with the arbitrary condi- a single ribozyme molecule could cleave multiple substrate tions used for the reactions shown in Figure 1. However, molecules. The was specific in cleaving a single ribozyme no reaction was observed in the absence of MgCl2, the same in phosphodiester bond, one as cleaved the indicating that neither spermidine nor KCl can replace natural cis-cleavage reaction. The trans-cleavage reaction magnesium in cleavage. exhibits Michaelis-Menten with 0.13 iM kinetics, Km and 0.7/min. Fedor and Uhlenbeck have kcat (1990) Effects of pH under single turnover conditions noted that kcat values in the range of 1/min and Km values The trans-cleavage reaction rate showed only a small pH in the nanomolar range are characteristic of many diverse dependence at equimolar concentrations of ribozyme and ribozymes. However, these measurements of kinetic para- substrate (Figure SE). However, these experiments were meters, including ours, used ribozymes removed from their performed at subsaturating concentrations of MgCl2 and natural sequence contexts and intracellular environment. they were probably not under single turnover conditions. Also, the rate-limiting steps may be different in the in in Consequently, it was possible that some step the reaction reactions of the different ribozymes. The similarities Km unless there is other than the actual cleavage step itself may have been and kcat may therefore be coincidental, the the effect of feature of that rate-limiting step, thereby masking some interesting but unrecognized biology increased hydroxide ion concentration. To investigate this results in selection for kinetic in steady-state parameters turnover conditions were established this possibility, single range. under reaction conditions The shortest region of VS RNA that func- empirically optimized by contiguous the initial rates of of 0.13 iM tions as a substrate in the trans reaction described here measuring trans-cleavage concentrations of contains a nucleotide of the site substrate by increasing ribozyme. Figure single upstream cleavage characteriza- 6A shows that the initial rate of increased with and 19 nucleotides downstream. Our cleavage previous showed that a concentration to -2.5 iM and tion of the reaction also only ribozyme up subsequently cis-cleavage of the site leveled that the reaction was nucleotide is off, suggesting approaching single required upstream cleavage in VS is similar to HDV turnover conditions. The rate as a function et this respect single cleavage (Guo al., 1993); which also a of concentration of MgCl2 was re-investigated using ribozymes, require only single upstream be the for cis- or and 0.13 iM S and Rz and found to nucleotide trans-cleavage (Perrotta Been, 5 ,uM essentially not a concentra- Based on minimum free same as in SF 1990, 1992, 1993). energy predic- shape Figure (data shown); and the aberrant tion of 25 mM MgCl2 was chosen to ensure that magnesium tions, electrophoretic mobility pattern we reactions 0.13 of to was not limiting. Trans-cleavage using ,uM accessibility single-strand-specific nucleases, substrate RNA consists of a of showed a minor conclude that the VS mostly S and Rz over a 5 ,uM range pH only structure flanked three nucleotides in reaction rate stable by enhancement (Figure 6B). stem-loop which be involved in 5' and 3' some of on the ends, may this structure reaction kinetics non-Watson-Crick (Figure 4B). Although Steady-state under the conditions of of it was structure to if Rz is appears predominate To determine capable multiple turnover, do not know that it is for molar excess of S the we necessary with an -20-fold reaction, incubated (Figure remains in this structure nor that the RNA molecule cleaved a If each cleavage, 7A). ribozyme only single of the effect the reaction. of 5% of S could be cleaved. In a maximum throughout Indeed, analysis substrate, 373 H.C.T.Guo and R.A.Collins B. A. C. 0. 1.2 0.16 1.0 __ Vmax _ .-f a1) 0.12 .' 0-0 a) E 8 0.6 6, -~0.' -.. O / E o 0'4 0.08 o.~ to. o-00 0.0.4- CIO > 0.04 0.13pM Km= 0 > 0.2 ) l o.. .. . . 0.0 0 0.02 0.04 0.06 0.08 0.2 0.4 0.6 0.8 time (min) [Rz] M [S] PM Fig. 7. Steady-state kinetics. Unless otherwise reactions were stated, carried out as described in Materials and methods at in mM 30°C Tris-HCl, pH 8.0, 5 mM 2 mM MgCl2, spermidine, 25 mM Rz is of KCl). (A) capable turnover. An -20-fold molar excess S multiple (-0.8 RM) was incubated with Rz (0.04 ,uM) and the fraction of substrate cleaved was determined at different times. Rate of is (B) cleavage proportional to ribozyme concentration in the presence of excess substrate. S was (0.75 incubated with 0.04 or 0.08 jM Rz ,uM) 0.01, and the initial rates 0.02, of cleavage were measured. (V0) (C) Determination of and Initial rates of Km of various concentrations of S Vmax. cleavage 0.01 jiM Rz by were determined. Substrate concentrations were 750 and 1000 nM. 2, 10, 50, 125, 250, was calculated as kcat divided V1max (0.14 pmol/min/20 pl) by [Rz] (0.2 pmol/20 ,ul). of base substitution mutations on In addition to in base cis-cleavage this interactions are pairing, tertiary stem-loop region (J.Olive and known or R.A.Collins, to contribute to unpublished suspected substrate binding by results) has revealed a of several complex pattern effects on et et ribozymes (Pyle al., 1992; Dib-Haij al., rate. cleavage Disruption of some base in the In pairs interactions alone stem 1993). fact, tertiary are sufficient to certain by single base substitutions has little or no allow weak > but effect very 0.1 of (Km mM) specific binding on cis-cleavage. However, at some the the of a positions P1 I intron to its identity stem-loop Group core catalytic of one of the bases in a is and particular pair (Doudna RNase P also critical; Szostak, 1989). recognizes even when the compensating substitution is made in substrates that the contain substantial secondary structure and complementary position to restore the have limited helix, cleavage is for Watson-Crick very potential with pairing not restored. We have just begun to the effect the study of et Westhof ribozyme (Smith al., 1992; and Altman, these mutations on trans-cleavage. Our current information We are more direct 1994). currently undertaking measure- suggests that specific bases at specific positions are more ments of the of and mutant binding wild-type substrates important than the to simply presence of a the idea stem-loop investigate that the high apparent affinity of structure. the VS for its ribozyme inferred from its substrate, low The stem-loop structure of the VS substrate RNA is due to interactions. Km (0.13 multiple gM), leaves no long regions available for The Watson-Crick pairing of the temperature optimum trans-cleavage reaction with the ribozyme. Our model for the is working structure lower than for the substantially cis-cleavage reaction of the ribozyme, inferred from the recently determined versus and (30 off much more -45°C) activity drops secondary structure of the minimal at cis-cleaving region of sharply higher temperatures (cf. Collins and Olive, VS RNA (see Figure 4C), predicts that the The ribozyme also retention of at 1993). activity higher temperatures in has no long single-stranded regions. This is in contrast the to cis-cleavage reaction indicates that the active site of most trans-acting ribozymes derived from the does hammerhead, ribozyme not to denature begin until at least hairpin, HDV and Group I intron RNAs, which have been 45°C. The lower optimum temperature of the trans- to designed interact with single-stranded regions of their reaction cleavage reflect decreased may binding of the substrates via formation of one or two intermolecular substrate at higher temperatures. helices flanking the site to be cleaved. In retrospect, it is We noted in our previous characterization of the VS fortunate that we constructed the trans RNA reaction in an reaction cis-cleavage that the cleavage rate was empirical fashion, rather than designing it based on know- unaffected essentially by pH (Collins and Olive, 1993). ledge of the secondary structure of VS RNA. Given the Consistent with this observation, the trans-cleavage precedents from most other ribozymes, it is unlikely that reaction described here also showed little, if any, pH we would have chosen to divide the VS sequence where even dependence, when examined under single turnover we did and expect the two RNAs to interact well. Indeed, conditions. These observations differ from results examin- after we determined the secondary structure of VS RNA, the ing rate of the chemical cleavage step of hammerhead we attempted to design a trans-reaction analogous to other ribozymes (Dahm and Uhlenbeck, 1993), RNase ribozymes, by dividing the VS sequence within loop I, (Guerrier-Takada et al., 1986; Smith and Pace, 1993; however, this combination of RNAs showed no activity Beebe and Fierke; 1994) and Tetrahymena Group I intron (A.Kawamura and R.A.Collins, unpublished results). This et (Herschlag al., 1993; Herschlag and Khosla, 1994). For could be due to inactive RNA conformations in the these the rate ribozymes, of the cleavage step was found ribozyme and/or substrate, or it may indicate that to increase with increasing pH. Failure to observe pH sequences in loop I are important for formation of the in dependence VS RNA could mean that OH- is not active structure. involved in the cleavage reaction, that the reaction 374 Trans-cleavage by a VS RNA ribozyme NaCl, 2 mM proceeds via a novel mechanism or, more likely, that the 300 U RNAguard spermidine-(HCl)3], (Pharmacia), 150- 200 U RNA T7 polymerase (Bethesda Research Laboratories) for 2 h VS trans reaction is not limited by the rate of the chemical at 37°C. Radioactive transcripts were prepared as above except an cleavage step under these conditions, but rather by some additional 30 jiCi [cx-32P]GTP (or, for specific experiments, ATP or step that precedes actual cleavage. UTP) was added. Samples were subsequently treated with DNase I (5 U/ jg DNA template; One interesting candidate for such a rate-limiting step Pharmacia) for 15 min, then EDTA was added to 10 mM. RNAs were extracted with would be a change in the substrate and/ phenol:chloroform:isoamyl alcohol, conformational chloroform:isoamyl alcohol (CIA) and in the ethanol-precipitated or nbozyme following binding. At saturating ribozyme presence of 0.3 M sodium acetate, pH 5.2. concentration, the pseudo-first-order rate constant for Precipitated RNAs were dissolved in water and two volumes of trans-cleavage of S (-0.6/min) is -10-fold higher than sequencing dye (95% formamide, 0.5X TBE, 0.1% xylene 0.1% cyanol, bromphenol blue), heated at the rate of cis-cleavage of RNA under similar 75°C for 3 min and fractionated GIl by electrophoresis on denaturing polyacrylamide gels (40:1 acrylamide:bis- conditions (determined from plots of fraction of uncleaved acrylamide) of appropriate 1 concentration containing 8.3 M urea and x RNA versus time of the data used for Figure 6 and Collins TBE (135 mM Tris, 45 mM boric mM RNAs were acid, 2.5 EDTA). and Olive, 1993). Since we envision that the trans- visualized either by autoradiography or UV Bands of interest shadowing. were excised, cleavage reaction recreates essentially the same RNA eluted overnight at 4°C in water and filtered to remove residual polyacrylamide. RNAs were precipitated with ethanol in the conformation as in the cis-cleavage reaction, the higher presence of 0.3 M sodium acetate and dissolved in water. Concentrations rate suggests that the cleavable conformation may be more were determined 1 spectrophotometrically, assuming corresponds OD260 easily attained when S (stem-loop I; Figure 4C) is not to an RNA concentration of 40 ,ug/ml. constrained by covalent attachment to the nbozyme core. End-labeling of RNAs In support of this idea, we have also found that the rate RNAs were labeled at 5' T4 kinase and termini using polynucleotide of cis-cleavage of GIl RNA can be increased several fold or at T4 RNA and End- 3' termini using ligase [y-32P]ATP 5'-[32P]pCp. by increasing the distance between stem-loop I and the labeled RNAs were on and fractionated denaturing polyacrylamide gels ribozyme core (A.Andersen and R.A.Collins, unpublished detected by autoradiography. In order to remove to 5' some results). These observations are consistent with the idea 5'-triphosphates prior end-labeling, RNAs were treated with U calf intestinal alkaline phosphatase of at least one conformational change involving the (Boehringer Mannheim) in a 10 p1 reaction 50mM containing Tris-HCI, substrate stem-loop occurring during the reaction. pH 8.0, 0.1 mM EDTA at 55°C for 30 min. Reactions were terminated The observation that the VS can recognize a ribozyme by extraction with phenol:CIA and CIA. substrate that contains a stable secondary structure may RNA sequencing be useful from the perspective of ribozyme engineering. End-labeled RNAs were partially digested with RNase TI or U2 Among the limitations to modifying hammerhead, hairpin essentially as described by Donis-Keller et al. in (1977) denaturing or Group I intron riboyzmes to cleave non-native target buffer (7 1 M urea, 50 mM citric acid, mM 0.05% EDTA, xylene cyanol, RNAs is the requirement that the target site be in a single- 150 yeast 5 jig/ml tRNA) and adjusted with NaOH to for TI pH digests and pH 3.5 for in ice. To stranded region to allow recognition via base pairing with U2. Reactions were terminated by chilling prepare partial RNA and 0.5 alkaline hydrolysis products, labeled the ribozyme. Because the cleavage site for the VS RIg/l yeast carrier tRNA for in 0.15 M were heated to 95°C 5-10 min NH40H, ribozyme is adjacent to a stable secondary structure, the lyophilized and dissolved in buffer. RNAs were denaturing separated by VS ribozyme may have unique properties that can be electrophoresis on 20% denaturing polyacrylamide gels. adapted to cleaving certain RNAs that are not accessible Structure probing to the action of other ribozymes. We are currently Limited partial digestion of 5' end-labeled S was under native performed investigating the substrate recognition requirements, conditions essentially as described Labeled RNA by Knapp (1989). (10- including whether substrate secondary structure is 100 ng) jl mM was dissolved in 27 of 50 mM 25 Tris-HCI, pH 8.0, required, or simply tolerated, by the ribozyme. tRNA. KCI, 10 mM MgC12, 2 mM 2.5 spermidine-(HCl)3, jg/jl yeast the reactions were RNase TI (4.5 U) or T2 (0.225 was added and U) incubated removed at 15 and at 30°C. Samples (3 jl) were 0.5, 2, 3, 8, and mixed Materials and methods 30 min (RNase T1) or 2, 3, 15 30 min 5, 8, (RNase T2), with an M 10% 0.05% equal volume of stop solution (9 urea, glycerol, DNA templates and synthesis of RNAs in ice. Products were to xylene cyanol) and frozen dry subjected 20% followed Fragments of VS DNA were cloned into vectors pTZ18R or 19R electrophoresis on denaturing polyacrylamide gels by (Pharmacia). Clone Gl (see Guo et al., 1993) contains VS nucleotides autoradiography. 617-881, numbered as in Saville and Collins (1990); the cleavage site is between G620 and A621. Substrate RNAs were transcribed (see Analysis of 5' and 3' termini nuclease below) from or its derivatives which had been linearized at the Identification of 5' and 3' terminal nucleotides P1 GlI by digestion labeled AvaI site (nucleotide 639) or the SspI site (nucleotide 783) to make and thin of two-dimensional layer chromatography appropriately RNAs designated GlIl/AvaI and GIl/SspI respectively. These RNAs RNAs was carried out as described previously 1967; (Shugar, Silberklang begin with nine vector nucleotides (5'-gggaaagcu) followed by VS et al., 1979; Saville and Collins, 1990). sequence. A site-directed mutant of G 11, clone 621 U, which contains a single A-*U substitution immediately following the self-cleavage site, Trans-cleavage reactions AvaI was carried out was also used. Trans-cleavage of S by the ribozyme (Rz) following X reaction S and Rz in the 1 Clone A-3 contains VS sequences downstream of the AvaI site pre-incubation of gel-purified appropriate addition of to Reactions were initiated (nucleotides. 640-881) in a derivative of pTZ19R that lacks the XbaI solution for 2 min. by ribozyme jl. In a 10 of in a final volume of 20 reaction, and SphI sites in the multiple cloning site (constructed for reasons substrate typical aliquots terminated addition of 13.5 jl unrelated to the project described here). Transcripts of clone A-3 digested 1.5 jl were removed at specified times, by 7 mM 0.07% with SspI (VS nucleotide 783) begin with 9 vector nucleotides (5'- of stop mix (70% EDTA, 0.4X TBE, formamide, xylene and stored on ice. were gggaaagcu) followed by 144 nucleotides of VS RNA; this RNA is cyanol, 0.07% bromphenol blue) Samples 20% on designated the AvaI ribozyme, or Rz. fractionated by electrophoresis gels. denaturing polyacrylamide and on The RNAs were prepared by in vitro T7 RNA polymerase transcription effects of temperature, pH, MgCl2 spermidine-(HCl)3 were the reaction from linearized plasmid DNAs. Transcription reactions (usually 300 jl) trans-cleavage incubating equimolar analyzed by jiM in solutions as described in concentrations of Rz and S contained icg appropriately linearized template, mM NTP (0.05 each) 10-20 1 each the effects of and For no (Pharmacia), 5 mM dithiothreitol, Ix T7 polymerase buffer [Bethesda the reason, NaCl KCl good figure legends. jM S. A final of the jM Rz and 0.125 at 0.05 Research Laboratories; 40 mM Tris-HCI, pH 8.0, 8mM MgCi2, 25 mM were examined study 375 H.C.T.Guo and R.A.Collins effects of MgCl2 under otherwise conditions was and 'optimized' Perrotta,A.T. Been,M.D. Nucleic Acids 3959-3965. performed (1993) Res., 21, at 30°C, 50 mM 2 mM 25 mM and Tris-HCI, pH 8.0, spermidine, KCI. Cech,T.R. 123-128. Pyle,A.M., Murphy,F.L. (1992) Nature, 358, Experiments to establish turnover conditions PhD single (Figure 7A) Saville,B.J. thesis, of Toronto. were (1991) University performed at in 50 mM 30°C 25 mM 25 and Tris-HCI, pH 7.1, mM Saville,B.J. Collins,R.A. Cell, 61, 685-696. (1990) MgC92, KCI, 2 mM spermidine. Analyses of the effect of under pH single Sharmeen,L., Kuo,M.Y.P., and J. Dinter-Gottleib,G. Taylor,J. (1988) turnover conditions (Figure 6B) were performed as 2674-2679. above, except that Virol., 62, the concentrations of Rz and S were 5 IM and 0.13 jM Methods respectively. Shugar,D. (1967) Enzymol., 12A, 131-137. Tris-HCI (50 was mM) used for pHs 7.1-8.9; 16.5 mM PIPES/44 and mM Silberklang,M., Gillum,A.M. Methods RajBhandary,U.L. (1979) Tris (Smith and Pace, was used for 1993) pH 6. 58-109. 19, Enzymol., Amounts of substrate and were products quantitated a and using Phosphor- Smith,D. Pace,N.R. (1993) Biochemistry, 5273-5281. 32, Imager and ImageQuant version 3.0 software (Molecular and Dynamics, Smith,D., Burgin,A.B., Haas,E.S Pace,N.R. (1992) J. Biol. Chem., Sunnyvale, CA). Estimates of initial rates cleavage were derived from 2429-2436. 267, plots of fraction of substrate cleaved versus time using Grafit software Symons,R.H. Annu. Rev. (1992) Biochem., 61, 641-671. (Erithacus Software Ltd, Staines, UK). Up to 90% of the substrate could Szostak,J. 83-86. (1986) Nature, 322, be cleaved in 60 min at concentration approximately equimolar of Uhlenbeck,O.C. (1987) Nature, 328, 596-600. ribozyme, with the curve indicating the presence of -10% and unreactive Westhof,E. Altman,S. Proc. Natl Acad. Sci. (1994) USA, 91, starting material. Curves were not adjusted to 100% and 5133-5137. completion the nature of the unreactive substrate has not been characterized further. Wu,H.-N, Wang,Y.-J., and Hung,C.-F., Lee,H.-J. Lai,M.M.C. (1992) J. 233-245. MoL Biol., 223, and Zaug,A.J. Cech,T.R. (1986) Science, 231, 470475. Ackcnowledaements Zuker,M. (1989) 48-52. Science, 244, We thank Murray Schnare for advice on ribonuclease sequencing, Anne Received on August 9, 1994; revised on October 1994 24, Kawamura for with help some of the single turnover experiments and Joan Olive for 3B. We Figure also thank Olke Debbie Uhlenbeck, Field, Diane De Abreu, Elisabeth Tillier and Jim Hogan for helpful discussions and Tom Cech for comments on the This work was manuscript. supported by an Operating Grant from the Medical Research Council of Canada. 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