A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes

CHAPTER THREE

A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes Timothy J. Wilson, David M.J. Lilley Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom

Contents 1. Introduction 2. The Hairpin and VS Ribozymes 2.1 The secondary structures of the hairpin and VS ribozymes—the role of helical junctions 2.2 Structure and dynamics of the hairpin ribozyme 2.3 Structure and dynamics of the VS ribozyme 2.4 Identification of the active site of the VS ribozyme 2.5 A new crystal structure of the VS ribozyme 2.6 The catalytic mechanism of the VS ribozyme 2.7 Cleavage and ligation reactions in the hairpin ribozyme—the internal equilibrium 2.8 Candidate catalytic components in the hairpin ribozyme 2.9 The catalytic mechanism of the hairpin ribozyme—is it same as that of the VS ribozyme? 3. General Themes Acknowledgments References

94 95 96 97 99 101 103 104 109 110 111 114 116 116

Abstract The hairpin and Varkud satellite ribozymes are two members of the class of nucleolytic ribozymes that catalyze cleavage and ligation reactions at a specific site. Cleavage occurs by a transesterification reaction whereby the 20 -O attacks the adjacent phosphorus with departure of the 50 -O to leave a 20 ,30 -cyclic phosphate. The structures of both ribozymes are now known. Although the sequences and structures of these ribozymes are generally unrelated, the topological organization and the arrangement of the active sites are the same for both. Both mechanistic and structural data indicate that the ribozymes employ general acid–base catalysis to accelerate the transesterification reactions, using guanine and adenine nucleobases as the general base and acid, respectively, in the

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cleavage reactions. As a class, the nucleolytic ribozymes all appear to use general acid– base catalysis; guanine nucleobases in particular are very common participants.

1. INTRODUCTION The hairpin1–3 and Varkud satellite (VS)4 ribozymes are members of the class of nucleolytic ribozymes that cleave or ligate RNA at a specific site within the ribozyme.5 The other members of this class are the hammerhead, hepatitis delta virus (HDV), and GlmS ribozymes. The biological role of these cleavage and ligation events is the processing of replication intermediates, except for the GlmS ribozyme which effects gene control by cleavage of mRNA.6 The nucleolytic ribozymes carry out cleavage at a specific site by a transesterification reaction in which the 20 -O attacks the adjacent phosphorus with concomitant departure of the 50 -O to leave a 20 ,30 -cyclic phosphate (Fig. 3.1). Ligation is simply the reverse reaction in which the 50 -O attacks the phosphorus with departure of the 20 -O to open the cyclic phosphate. The reactions follow an SN2 mechanism with inversion of configuration at the phosphate. The reactions are accelerated by at least 105-fold when

B1

B1 O O

O

O

H

P O

O

P Y

H

H

O

O

:X

cleavage O

P O

B2 O O

O

O

O

ligation

B2

OH

X

O O

O

O O

B1

O

Y:

H

B2

O O

OH

O

OH

Figure 3.1 The proposed chemical mechanism of nucleolytic ribozymes cleavage by general acid–base catalysis. The transesterification reaction proceeds by attack of the 20 -O nucleophile to generated a cyclic 20 ,30 -cyclic phosphate in the cleavage reaction (highlighted red), or by attack of the 50 -O in the reverse ligation reaction (blue). In the cleavage reaction, the 20 -O nucleophile is deprotonated by a general base, and the 50 -O leaving group is protonated by a general acid. In the ligation reaction, the roles of general base and acid are reversed to deprotonate the 50 -O nucleophile and protonate the 20 -O leaving group.

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catalyzed by the ribozymes, although significantly greater rate enhancements have been demonstrated recently for specific forms of the hammerhead and VS ribozymes.7,8 Understanding just how RNA molecules can generate significant acceleration of these phosphoryl transfer reactions is a major goal. Inspection of the chemical mechanism (Fig. 3.1) suggests a number of possible strategies. Many enzymes lower activation barriers by differential stabilization of the transition state structure. For the nucleolytic ribozymes, the transition state will strongly resemble the pentacoordinate phosphorane, which might be stabilized by hydrogen bonding or juxtaposition of positive charge. The reaction requires the nucleophile to attack in-line with the 50 -O leaving group, and if the structure can facilitate this trajectory it might provide a rate enhancement of perhaps 10–100-fold. Looking at the protein world suggests another possible source of catalysis. The enzyme pancreatic ribonuclease A catalyzes essentially the same reaction as the nucleolytic ribozymes using general acid–base catalysis. A general base is used to deprotonate the attacking nucleophile; an alkoxide ion is a stronger nucleophile than a hydroxyl by many orders of magnitude. In parallel, a general acid is used to protonate the oxyanion leaving group, facilitating its departure. In RNase A, the imidazole side chains of two histidine residues provide the general acid and base. These residues are ideally suited to the task because they have pKa values close to physiological pH. By contrast, RNA is limited to nucleobases, hydroxyl groups, and hydrated metal ions, all of which have pKa values that are significantly shifted from physiological pH. This lowers the effective concentration of active catalyst. For example, if a general acid has a pKa of 5 then only 1% will be protonated at neutral pH. However, the low effective concentration of acid will be offset to some degree by its greater reactivity. The observed rates for nucleolytic ribozymes are generally slow, but turnover is not required for their biological function.

2. THE HAIRPIN AND VS RIBOZYMES Both the hairpin and VS ribozymes process replication products that are transcribed from RNA and DNA, respectively. The tobacco ringspot virus has a circular single-stranded satellite RNA of 359 nt. In its replication cycle, the negative strand of the satellite RNA is produced as a concatameric transcript that is processed into monomeric circular molecules by sequential cleavage and ligation reactions catalyzed by the hairpin ribozyme within the RNA. The VS RNA is an abundant transcript from a plasmid found in the

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mitochondria of some field-isolated strains of the fungus Neurospora crassa from Varkud in Kerala, India. Collins and coworkers found that the VS RNA contains an element capable of self-cleavage,9 which serves a role in the processing of replication intermediates.10

2.1. The secondary structures of the hairpin and VS ribozymes—the role of helical junctions The nucleolytic ribozymes are relatively small, autonomously folding RNA species. Two members of this class, the HDV and GlmS ribozymes, have folds based on complex, nested pseudoknots, whereas the remaining members have one or more helical junctions. There appears to be alternative solutions to the problem of generating a stable fold that permits catalysis, and based on this rather small set of ribozymes, the solutions seem to be mutually exclusive. The secondary structure of the hairpin ribozyme shows it to be centered on a four-way helical junction, with arms sequentially labeled A, B, C, and D (Fig. 3.2).3 Adjacent arms A and B contain loops that include all but one of the nucleotides shown to be essential for catalytic activity, and the site of cleavage/ligation is located in arm A. Much of the initial work on this ribozyme was carried out in the Burke lab14–18 studying a minimal form comprising only helices A and B connected by a single-stranded hinge. The sequence and deduced secondary structure of the VS ribozyme19 suggests that it too should be based upon helical junctions (Fig. 3.3). Two three-way junctions interconnect five helical sections (helices II through VI) forming a nominal H shape. Cleavage and ligation reactions occur within the internal loop of stem-loop I that is connected to the end of helix II. Collins and coworkers showed that the terminal loop of helix I interacts with that of helix V,23 and they have suggested that this results in an altered pattern of base pairing that propagates down the helix to change the structure within the internal loop that contains the scissile phosphate.24 This involves the opening of the terminal base pair, extrusion of C634, and rearrangement of base pairing in stem I such that CCC (635–637) pairs with GGG (623–625). Jones and Strobel25 pointed out that an additional helix (VII) in the natural VS RNA sequence might play a role in the function of the ribozyme by forming a third three-way junction with helices I and II. Helix I may be physically disconnected from the rest of the ribozyme, such that a ribozyme comprising helices II through VI acts in trans upon stem-loop I.26 Helix I may also be reconnected to the ribozyme in a variety

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Figure 3.2 The sequence and secondary structure of the hairpin ribozyme. The ribozyme is based upon a perfect (4H) four-way helical junction with arms labeled A through D. Arms A and B contain internal loops that interact to generate the active site of the ribozyme. The site of cleavage within loop A is indicated by the arrow. All the critical nucleotides required for catalytic activity are located in the two loops, including G8 within loop A and A38 within loop B, and the two loops make an intimate interaction in the folded ribozyme.11–13

of different ways with retention of cleavage activity, and in some cases with markedly enhanced rates.8

2.2. Structure and dynamics of the hairpin ribozyme The location of the nucleotides critical to ribozyme activity in the loops of the A and B helices led to a strong suspicion that the active form of the ribozyme would be generated by an intimate interaction between the loops. The interaction would probably remodel the structures of both loops and create the chemical environment in which catalysis of the transesterification reaction could occur. However, the minimal hinged form of the ribozyme required unphysiologically high concentrations of divalent ions to facilitate catalysis, and attention turned back to the form with an intact four-way

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Figure 3.3 The sequence and secondary structure of the VS ribozyme.19 The proposed secondary structure comprises seven helical segments (labeled I through VII) connected by three three-way helical junctions. Helix I can be considered as the substrate and it can be physically disconnected from a trans-acting ribozyme consisting of helices II through VI. Cleavage and ligation reactions take place within the internal loop of helix I at the position indicated by an arrow. Two critical nucleotides for catalytic activity are A756 (within the A730 loop of helix VI),20,21 and G638 within the internal loop of helix I.22

junction. FRET studies with this species showed that the junction folds by coaxial stacking of helices A on D and B on C in the presence of metal ions,11 reminiscent of the folding of the four-way DNA junction.27 Indeed, the four-way junction in isolation (i.e., with the loops removed) predominantly folds into the same stacking conformer as the complete ribozyme, so that the junction predisposes the ribozyme to fold correctly.28 These studies provided the first physical evidence for loop–loop interaction. A similar analysis was performed using the hinged form.29 Single-molecule FRET studies of both the hinged30 and four-way junction31 forms of the ribozyme revealed metal ion–dependent dynamics, with repeated docking and undocking of the loops. The folding of the four-way junction form occurs in two stages, with a fast ( 100 s1) scissor-like rotation of the junction that presents the loops to each other, and a slower (3 s1) docking process.31 The rate of docking in this form is about 500 times faster than that measured in the hinged form.30 Therefore, folding is not normally rate limiting in the junction form of the ribozyme. The four-way junction is not required for catalytic activity, but it acts as an auxiliary element that assists the folding of the ribozyme under physiological conditions12,13,28 similar to the loops of the hammerhead ribozyme.32,33

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Figure 3.4 Parallel-eye stereo image of the crystal structure of the hairpin ribozyme in the full four-way junction form.34 The ribozyme folds by the coaxial stacking of helices A with D (magenta) and B with C (cyan). Note the close association between the internal loops of helices A and B. This includes the extrusion of Gþ1 from loop A and insertion into loop B where it base pairs with C25 (shown as sticks).

Crystal structures have been solved for both the junction34 (Fig. 3.4) and minimal hinged form35 of the hairpin ribozyme. These confirmed the key feature of the loop–loop interaction, and the general folding principle of the junction form. The loop–loop association involves an extensive set of contacts that include A-minor interactions, nucleobase contacts, and the extrusion of G þ 1 (one of the nucleotides that flank the scissile phosphate) from the A-loop and insertion into a pocket in the B-loop where it makes a Watson–Crick pairing with C25.34 The core nucleotides of the junction and hinged forms can be superimposed with an RMS deviation of ˚ .35 As expected, the intimate interaction between the loops alters 1.28 A the conformation of each loop from that of the isolated helices.36,37 The structure of a derivative of the hairpin ribozyme with a pentacoordinate vanadium replacing the scissile phosphate as a transition state model was obtained in the junction form,38 and this will be discussed in the following section.

2.3. Structure and dynamics of the VS ribozyme Unlike the hairpin ribozyme, until very recently there was no crystal structure for the VS ribozyme. Nevertheless, we already had good idea of the general fold of the ribozyme at low resolution from biophysical studies. Initial biophysical studies focused on the II–III–VI and III–IV–V junctions using a combination of comparative gel electrophoresis, FRET, and homology-based modeling.39,40 This suggested a core structure based upon

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the approximate coaxial alignment of helices IV, III, and VI. It was proposed that the probable location of helix I was the cleft formed between helices II and VI such that it could be connected to helix II and make a loop–loop interaction with helix V.40 Helix I of the substrate protrudes unhindered from the complex and can be extended without loss of catalytic activity.41 Both strands of helix II adjacent to the II–III–VI junction were found to be protected against hydroxyl radical attack upon folding42; a result consistent with a close association between the substrate and helix II in this region. The interaction might be mediated by 20 -hydroxyl groups on the substrate and helix II.43 nucleotide analog interference mapping (NAIM) experiments revealed that the removal of 20 -hydroxyl groups at the junction-proximal end of helix II was deleterious,44 and the formation of an A-minor interaction was suggested.45 The isolated junctions are induced to fold by the addition of divalent metal ions. The folding transitions are two-state in response to the noncooperative binding of counterions.39,40 There is no evidence for a requirement for site-specific binding of metal ions in the folding of the ribozyme junctions. In the absence of a crystal structure, we turned to small-angle X-ray scattering (SAXS) in solution to study the component junctions of the transacting form (helices II–VI) and the complete ribozyme comprising helices I through VII.46 Radii of gyration and maximum chord length measurements confirmed the probable codirectional orientation of helices IV–III– VI. Reconstructions based on the full scattering curves generated a family of closely related structures. The overall electron density envelope was rather flat, with helix-width protrusions at three corners, and a helical ridge running across the face. These features enabled us to assign the features of the low-resolution electron density envelope to the known helical sections of the ribozyme. The substrate helix I has been the subject of several studies by NMR. In one study, the upper stem-loop of helix I was truncated and terminated with a tetraloop,47 whereas a complete substrate strand was analyzed in another study.48 In both structures, the internal loop comprised two sheared G•A base pairs and a protonated Aþ•C pair, the former being similar to domain 2 of the hammerhead ribozyme.49,50 In a different study,51 the sequence was changed to force the proposed conformation when the terminal loop interacts with that of helix V.24 The sheared G620•A639 pair is preserved, but G638 interacts with both A621 and A622 in a noncoplanar manner, while A621 is cross-strand stacked onto A639 on the minor groove side.

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It might be expected that the VS ribozyme would be a dynamic structure in solution, but there is less direct information on this compared to the hairpin ribozyme. The folding of the complete ribozyme could be followed by its compaction (revealed as a reduction of radius of gyration) with an increase of Mg2þ ionic concentration, fitting to a two-state model with a [Mg2þ]1/2 of 330 mM and a near-unit Hill coefficient.46 However, the transition is not complete with less than millimolar concentrations of Mg2þ, and the best X-ray scattering profiles were obtained in the presence of high metal ion concentration. We think that the transverse ridge in the structure is occupied by helix I, but it is not certain to be 100% occupancy. The interaction with helix I is not especially strong; kinetic analysis of the cleavage of helix I by 20 ribozyme in trans gives an apparent affinity of Kapp SingleM ¼ 1 mM. molecule FRET experiments using ribozyme (lacking helix VII) carrying fluorophores on the 50 termini of helices I and VI indicated a dynamic structure in the presence of 50 mM Kþ and 35 mM Mg2þ. The results were interpreted to indicate that one-third of the species had undocked substrate at any given time in these conditions.52 Taken together, the data suggest that there is probably a reversible association between the substrate helix I and the core of the VS ribozyme that is similar to the hairpin ribozyme. Furthermore, it has been suggested that in vivo the ribozyme could act on a more distant substrate stem-loop in cis.53 Consistent with these ideas, we demonstrated a trans cleavage reaction between two complete VS ribozymes.54 This reaction clearly requires that helix I of one ribozyme must be able to displace that of another ribozyme and dock in its place. From these experiments, we estimated Kapp M ¼ 1.7 mM.

2.4. Identification of the active site of the VS ribozyme The key catalytic components of the VS ribozyme have been identified by nucleotide substitution. Most sequence changes in the ribozyme that affect catalytic activity do so because they alter the structure. These are typically located in the helical junctions or the bulges.39,40 Changes in the lengths of critical helices III and V are also deleterious because they affect the relative orientation of helices II and V or the loop–loop interaction between helices I and V, respectively.40 However, sequence changes in the internal loop of helix VI, termed the A730 loop, have no discernible effect on folding, yet most single-base changes introduced into this loop result in significant loss of cleavage activity (generally 50-fold or more) in trans20 and in cis.55 The A730 loop had earlier been found to be sensitive to ethylation interference, and it

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contained sites of interference by phosphorothioate incorporation and suppression by thiophilic metal ions.56 Sequence changes in the loop also affected ligation activity,41 and the loop was also very sensitive to the introduction of nucleotide analogs.45 In our SAXS-derived model of the ribozyme,46 the scissile phosphate is readily juxtaposed with the A730 loop. Docking the substrate into the cleft between helices II and VI naturally facilitates a close interaction between the cleavage site and the A730 loop. Considering the major sensitivity of ribozyme activity to changes in this region, the results suggest that the A730 loop is likely to be a significant part of the active site of the ribozyme. Within the A730 loop, one particular nucleotide stands out. Substitution of A756 by G, C, or U leads to 300-fold loss of cleavage20 and ligation activity.41 These changes had only a small effect on substrate binding affinity (△Kd  fivefold), and most of the effect arose from a reduced rate of central conversion of the substrate into product.20 In NAIM experiments studying ligation rate, the 756 position was the most sensitive nucleotide to substitution by a variety of analogs.45 UV cross-linking data placed A756 physically close to the cleavage site in the substrate.57 Removal of the 20 -hydroxyl group of A756 resulted in a small reduction in observed cleavage rate, whereas removal of the nucleobase (creating an abasic site at position 756) lowered the activity 1000-fold. Removal of the exocyclic amine from the 6 position, translocation to the 2 position, or addition of a 2-NH2 group all led to 1000-fold loss of cleavage. By contrast, replacement of N7 by CH (7-deazaadenosine) had a negligible effect on activity. Thus, the Watson–Crick edge of the nucleobase of A756 is important for catalytic activity. While activity of the A756 abasic VS ribozyme could not be restored by high concentrations of imidazole in the medium,21 a variant ribozyme with a covalently linked imidazole at position 756 demonstrated significant rates of cleavage and ligation.58 The activity of the A756 imidazole ribozyme suggested that A756 might be involved in general acid–base catalysis. This was also supported by other substitution experiments at this position.45 Consequently, we thought it likely that A756 might have a partner in general acid–base catalysis that could be another nucleobase somewhere in the RNA. The nucleobase was unlikely to be located within the main body of the ribozyme, but we identified G638 as a strong candidate for the role.22 Substitution of G638 with any other nucleotide resulted in a reduction of cleavage rate by four orders of magnitude, whereas substrate folding was not perturbed. The variant substrates bound the ribozyme with similar affinity, acting as competitive

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inhibitors of the natural sequence substrate. In other words, the G638A substrate bound to the ribozyme in an apparently normal manner, but was catalytically incompetent. This strongly suggested that G638 plays a direct role in the function of the ribozyme. Since its 20 -hydroxyl is dispensable,59 it is probable that the guanine nucleobase is the key participant, and nucleotide substitution experiments revealed that the key functionalities lie on the Watson–Crick edge.22 We also explored the cleavage of a substrate in which G638 was replaced by an imidazole nucleoside. Initially, we used the same compound that has been shown to be active when replacing A756,58 but this was found to be inactive.22 However, in this species, the imidazole ring is bonded directly to the C10 atom of the ribose (C0-linked imidazole), so that it is not possible to superimpose either ring N with N1 of guanine. We therefore made a new derivative in which two methylene carbon atoms link imidazole to ribose (C2-linked imidazole), and which exhibited measurable activity60 and provided further evidence supporting general acid–base catalysis. These studies collectively point to an involvement of two key nucleobases, A756 and G638 in the catalytic mechanism of the VS ribozyme. An intimate association of the A730 loop and the internal loop of substrate helix I would bring these nucleotides and the scissile phosphate into juxtaposition. In the NMR structure of the substrate RNA,51 N1 of ˚ from the 20 -OH group of G620 (the nucleophile). We suspect G638 is 6 A that the loop–loop interaction with the A730 loop leads to structural reorganization that generates the active conformation.

2.5. A new crystal structure of the VS ribozyme A crystal structure of the VS ribozyme was solved that confirms many of the features described above (Suslov, N., Huang, H., Lilley, D. M. J., Rice, P., and Piccirilli, J. A., unpublished data). The structure has a resolution of ˚ in the best direction and the electron density is readily interpretable 3.07 A for the whole molecule. The first point to note is that in the crystal the RNA had dimerized in exactly the manner we had deduced from our earlier complementation experiments.54 Thus the substrate stem-loop I of one ribozyme molecule docked into the cleft between helices VI and II of the other in a mutual manner like the number 69. The junction of helices III, IV, and V was almost exactly as previously deduced,39 although the junction of helices II, III, and VI was slightly different from our earlier model40 in that helices II and III are coaxial. The loop I–loop V interaction occurs as

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expected by Watson–Crick base pairing.23 Our earlier SAXS data clearly showed that the ribozyme can be monomeric in solution46 (and indeed a monomer taken from the crystal structure fits with the electron density envelope calculated from SAXS data) so that it is probable that there is a monomer–dimer equilibrium in solution. The active site of the dimeric ribozyme is clearly seen in the crystal. G620 and A621 flanking the scissile phosphate are splayed apart and are stacked with G638 and A756, respectively. Thus, these nucleobases are positioned consistent with their proposed roles as general base and acid, respectively, in the cleavage reaction. For this, they would need to move a little closer to form the active state, but there is nothing that would seem to prevent this. Thus the crystal structure is in excellent agreement with our earlier mechanistic studies.

2.6. The catalytic mechanism of the VS ribozyme There are a number of possible roles for A756 and G638 in the generation of the catalytic rate enhancement. They might stabilize the transition state by hydrogen bonding the phosphorane, or a positively-charged protonated adenine base might provide electrostatic stabilization of the dianionic transition state. However, the available evidence points toward general acid– base catalysis by these nucleobases. A chemical mechanism consistent with the roles of G638 and A756 in general acid–base catalysis is shown in Fig. 3.5.22 Clearly, this catalytic B

A GUA

H2N

N

O

N

GUA

N

O

N O

O

N

H

O

O O

P

O

O

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N

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O

N

N

NH2 N

N

H H2N

H

O O

O

O

N

N

P

ADE

H

N

N

O

OH

OH

O N N

Figure 3.5 Two alternative mechanisms for cleavage of the hairpin and VS ribozymes based on general acid–base catalysis by adenine (A38 and A756, respectively) and guanine (G8 and G638) nucleobases. (A) Deprotonated guanine acts as general base to deprotonate the 20 -O, and protonated adenine protonates the 50 -O leaving group. (B) Neutral adenine acts as general base to deprotonate the 20 -O, and neutral guanine protonates the 50 -O leaving group.

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strategy requires that the acid be protonated and the base unprotonated at the outset of the reaction. The observed rate of reaction (kobs) will be given by kobs ¼ kcat  fA  fB where fA and fB are the fractions of protonated acid and unprotonated base, respectively, and kcat is the rate of cleavage catalyzed by the ribozyme in the requisite state of protonation and is independent of pH. fA and fB can be calculated for any pH if pKa values are assumed, and thus the pH dependence of cleavage rate can be simulated.61 We reinvestigated the pH dependence of the cleavage reaction in the presence of a high concentration of Mg2þ ions, and obtained a bell-shaped pH dependence (Fig. 3.6), which was fitted to a double-ionization model with apparent pKa values of 5.2 and 8.4.22 The lower value is very much in agreement with an adenine in an electronegative environment, while the upper value is consistent with a guanine base if the pKa were reduced by proximity to metal ions. Using the fast-cleaving cisacting form of the ribozyme, Smith and Collins62 also obtained a bell-shaped pH dependence for cleavage, with pKa values of 5.8 and 8.3. We have carried out a detailed analysis of pH effects for position 638 using the trans cleavage reaction. A G638I substrate produced a bell-shaped 8 7 6

kobs

/ min-1

5 4 3 2 1 0 4

5

6

7 pH

8

9

10

Figure 3.6 pH dependence of the rate of substrate cleavage by the VS ribozyme.22 The data have been fitted to a double-ionization model.

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cleavage rate pH profile, with an upper pKa reduced by 0.3 units,22 where the expected pKa of inosine is 0.5 unit lower than that of guanine. Interestingly, the lower pKa was also reduced by 0.4 unit, suggesting that A756 is close enough to G638 to be influenced by the substitution. When our C2linked imidazole nucleoside60 was placed at this position, we obtained an upper pKa of 7.0 (unpublished data). Substitution of G638 by 2,6diaminopurine (DAP) with a normal pKa of 5.1 produced a rate of cleavage that was reduced 1000-fold relative to the natural sequence at pH 8. However, it significantly restored activity at lower pH with a corresponding pKa of 5.6. The resulting rate was log-linear with pH over the range from 6 to 8 with a unit gradient. This indicates that general acid–base catalysis contributes over two orders of magnitude to the catalytic power of the VS ribozyme. The pH dependence of a reaction may reflect a change in the ratelimiting step rather than the protonation state of reactants, but several lines of evidence suggest that this is not the case for the VS ribozyme. Kinetic isotope effects in a fast, cis-acting form of the VS ribozyme show that proton transfer occurs in the transition state of the cleavage reaction.62 In the trans form of the ribozyme, the central conversion of substrate to product is rate limiting, with rapid and pH-independent substrate binding.22 Finally, the correlation between the pKa of the nucleobase at position 638 and the observed pKa of the cleavage reaction strongly suggests that the rate of reaction depends on protonation state of the nucleobase. While a substantial part of the VS ribozyme rate enhancement may result from general acid–base catalysis by A756 and G638, the pH profiles do not allow us to determine which nucleobase is the acid, and which the base. The two alternatives lead to identical predicted pH dependence. A similar ambiguity existed for the HDV ribozyme, but was resolved by the use of a 50 -phosphorothiolate (50 -PS) substitution at the scissile phosphate.63 The 50 -S atom is a much better leaving group than the normal O, thus not requiring protonation. As a consequence, substitutions in the ribozyme that impair the function of the general acid and lower the activity on the oxy (50 -PO) substrate should have little effect on cleavage of a 50 -PS-containing substrate. In other words, the 50 -PS substitution should “rescue” the cleavage activity of ribozyme in which the general acid has been removed. On the other hand, impairment due to changes in the general base will not be rescued by 50 -PS substitution. In an analogous experiment, it was previously shown that the cleavage of uridine 30 -(p-nitrophenyl phosphate) was unaffected by an H119A mutation in ribonuclease A64 because the p-nitrophenyl

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Subtrate +

Ribozyme A756

None

O

S

O

A756G

S

O

S

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Figure 3.7 Cleavage of the VS substrate by A756 or A756G ribozymes as a function of the presence or absence of a 50 -phosphorothiolate (PS) linkage at the scissile phosphate.65 The 50 -O (O) and 50 -PS (S) substrates were incubated with no ribozyme (none), natural sequence ribozyme (A756), or ribozyme in which the critical A756 is replaced by G (A756G) for 15 min, and the products of cleavage were separated by gel electrophoresis. Note that cleavage of the oxy substrate is strongly impaired when the A756G ribozyme is used, but that cleavage is restored by the phosphorothiolate substitution. This is strongly indicative of A756 acting as general acid in the cleavage reaction.

is such a good leaving group that it does not require protonation by a general acid. However, an H12 mutation was not rescued, consistent with its function as a general base activating the nucleophile. We found that the cleavage activity of VS A756G ribozyme was impaired 1000-fold acting on the oxy (50 -PO) substrate, but the activity was completely restored with the 50 -PS-containing substrate (Fig. 3.7).65 The cleavage of the 50 -PS substrate thus became insensitive to substitution at position 756, and we concluded that A756 is therefore the acid. By contrast, the rate of cleavage of a G638DAP plus 50 -PS substrate was similar to that observed for a G638DAP plus 50 -PO substrate, and both were significantly lower than the natural sequence. The pH profile of cleavage rate for the G638DAP plus 50 -PO substrate is bell-shaped, with pKa values of 4.8 and 5.6.22 However, with the 50 -PS substitution the profile changed, with the reaction rate increasing to pH 6 and remaining at a plateau thereafter.65 The rate would not be expected to fall at higher pH, if deprotonation of the acid is no longer relevant. These data were fitted to a single ionization, with a pKa of 5.3, consistent with general base catalysis by the diaminopurine at position 638.

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All the available data are therefore consistent with a catalytic mechanism for the VS ribozyme cleavage reaction in which G638 acts as general base to deprotonate the 20 -O nucleophile, and A756 is the general acid protonating the 50 -oxyanion leaving group. By the principle of microscopic reversibility, in the ligation reaction a protonated G638 should act as the general acid protonating the 20 -oxyanion leaving group, and an unprotonated A756 as general base deprotonating the 50 -O nucleophile that attacks the cyclic phosphate. Having assigned the acid and the base, it is interesting to compare the VS ribozyme to its protein equivalent RNase A. Given pKa values of 5.2 and 8.4 for the ribozyme, the product fA  fB achieves a maximum of 3.8  104 and thus kcat is 330 s1 under the conditions of the assay. This is not much lower than the kcat for RNase acting on various substrates, of 1400 s1.66 Of course, the superiority of the protein enzyme lies in the maximum fA  fB of 0.25. Is this the complete story of how the VS ribozyme reaction is catalyzed, or could other components to the overall rate enhancement exist? The direct participation of a site-bound metal ion, either as a Lewis acid or in general acid–base catalysis, is unlikely because the ribozyme is active in monovalent ions.67 However, some electrostatic stabilization of the transition state by nonspecifically bound ions may occur. It is possible that the transition state might also be stabilized by interactions between the key nucleobases and the phosphorane, as suggested by the crystal structure of the hairpin ribozyme (see below).38 Alternatively, a protonated adenine (other than A756) might stabilize the dianionic phosphorane, as proposed for the hairpin ribozyme.68 However, these effects seem unlikely to be major contributors to the ribozyme rate enhancement because they should not be reversed by activation of the leaving group with 50 -PS substitution. If the sole function of the ribozyme is to present A756 so that it can transfer a proton in the transition state, then the ribozyme might be unnecessary for the cleavage of the 50 -PS substrate. While the cleavage rate of the natural sequence (i.e., G638 unmodified) 50 -PS substrate in the absence of ribozyme was found to be 750-fold faster than the oxy form, the rate was nevertheless increased an additional 70-fold by the addition of a trans-acting ribozyme.65 Interaction of the substrate with the ribozyme probably remodels the structure of the substrate into a conformation required for full activity. This might bring the nucleobase of G638 closer to the 20 -OH group of G620. This would also be anticipated to reposition the 20 -O for in-line nucleophilic attack, which should generate a further modest rate enhancement.

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2.7. Cleavage and ligation reactions in the hairpin ribozyme—the internal equilibrium The four-way helical junction of the hairpin ribozyme is conformationally predisposed to fold into the conformer that juxtaposes the loops of the A and B arms,28,31 allowing the ribozyme to fold in physiological ionic conditions.11–13,31,69–71 It also promotes the ligation reaction.72 FRET has been used to follow the cleavage and ligation reactions in the individual hairpin ribozyme.73 Single ribozyme molecules can be observed switching between two distinct dynamic modes (Fig. 3.8). In one state (assigned as the ligated species), the molecule remains stably docked for a period before changing into another form (assigned as the cleaved form) that undergoes rapid docking and undocking. The docking and undocking rates within the cleaved

cle

cleave

undock

ligate

dock

lig

cle

lig

cle

lig

EFRET

1.0 0.5 0 0

20

40

60

80

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Figure 3.8 Cleavage and ligation reactions observed in single hairpin ribozyme molecules.73 Junction-form hairpin ribozymes fluorescently labeled at the termini of the A and B arms were tethered to the surface of a quartz slide, and FRET between donor and acceptor was measured from individual ribozyme molecules over a period of time. FRET efficiency is plotted as a function of time for one molecule. The molecule can be seen to switch between two states. In one state (assigned as the ligated form), the molecule is stably folded into a structure that places the fluorophores close together (and hence high FRET efficiency EFRET is observed). In another state (assigned as the cleaved state), there is rapid oscillation between a docked state (high EFRET) and a more extended structure where the loops are not associated (low EFRET). The interpretation is shown by the schematic above the time trace. The transition from stable to oscillating states is deduced to be a cleavage event (cle), and the transition from oscillating to stable states is assigned as a ligation event (lig). From the duration of the ligated and cleaved states, the kinetics of cleavage and ligation reactions can be calculated.

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1 1 state are kC and kC dock ¼ 2.5 ( 0.1) s undock ¼ 2.3 ( 0.1) s ; these rates are significantly slower than the junction dynamics under the same conditions.31 Data from many single molecules give the internal conversion rates for the hairpin ribozyme. The cleavage rate (kC) is 0.6 min1 in the presence of 1 mM Mg2þ, while the ligation rate (kL: the rate of ligation in the docked ribozyme) is 0.3 s1. Thus the reaction is biased toward ligation, with an internal equilibrium constant of Kint ¼ kL/kC ¼ 34. The hinged form of the ribozyme is similarly biased toward ligation, but overall the cleavage rate is limited by docking and undocking rates.74 The bias to ligation maintains the integrity of the circular () strand allowing it to act as a template for (þ) strand synthesis. In the junction form, however, cleavage of the concatenated product of replication is facilitated by the rapid undocking that follows cleavage. Thus, the four-way junction confers the properties required for the biological function of the ribozyme.

2.8. Candidate catalytic components in the hairpin ribozyme Like the VS ribozyme, there is no evidence for a direct role of site-bound metal ions in catalysis. Significant levels of activity can be maintained in both ribozymes in the presence of high concentrations of monovalent ions such as 67 Liþ and NHþ The hairpin ribozyme was also shown to remain active 4. when Mg2þ ions were replaced by substitutionally inert hexamminecobalt (III) ions75–77 or aminoglycoside antibiotics.78 This excludes a requirement for a site-bound metal ion acting as a Lewis acid to activate nucleophilic attack, or ion-bound water molecules performing general acid–base catalysis. In addition, cleavage rates measured in single-molecule experiments were found to be almost independent of Mg2þ ion concentration.73 There is, however, evidence for nucleobase involvement in catalysis by the hairpin ribozyme. The first to be suggested was G8, located on the opposite strand from the scissile phosphate in loop A. Substitution by other nucleotides led to rates of cleavage being reduced by two orders of magnitude in both the hinged form79–81 and the junction form of the ribozyme,82 without significantly affecting ion-induced folding in the latter. Complete ablation of the nucleobase to leave an abasic site also led to a major loss of ribozyme activity.83 The cleavage rate was found to decrease with pH when DAP was substituted at this position, showing that catalysis was dependent on proton transfer involving a group with a pKa of 6.9.80 We introduced the C0-linked imidazole nucleoside84 at position 8.85 The substitution did not perturb folding, and the modified ribozyme was active in both cleavage

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and ligation at rates 10-fold faster than that for the G8U variant. Physical evidence for the proximity of G8 to the active center of the ribozyme was provided by Thomas and Perrin,86 who found a transfer of the alkyl group to G8 from bromoacetamide attached at the position of the 20 -OH nucleophile. The proximity of G8 to the catalytic center was shown crystallographically in a structure of the ribozyme in which the scissile phosphate was replaced by vanadate as a model of a pentacoordinate phosphorane transition state (Fig. 3.9).38 G8 was found to be hydrogen bonded to the 20 -O and the proS nonbridging O of the scissile phosphate, and was well positioned to participate in the catalytic chemistry. The structure also revealed the presence of a second nucleotide juxtaposed with the scissile phosphate. The nucleobase of A38 (contributed by loop B) was found to form hydrogen bonds to the 50 -O and the proR O. Removal of this nucleobase resulted in a 10,000-fold loss of activity,87 while ligation activity was shown to be sensitive to substitution by inclusion of modified nucleotides in NAIM experiments.88

2.9. The catalytic mechanism of the hairpin ribozyme—is it same as that of the VS ribozyme? There is a striking similarity between the active sites of the VS and hairpin ribozymes in our current view. Both are formed through the interaction of

Figure 3.9 Parallel-eye stereo image of the crystal structure of the active site region of a transition state analog of the hairpin ribozyme.38 The ApG segment containing the reaction site is highlighted green, but in this structure the phosphate is replaced by a vanadium atom to mimic a pentacovalent phosphorane transition state. Note that G8 is hydrogen bonded to the 20 -O and A38 is hydrogen bonded to the 50 -O, consistent with the proposed mechanism of general acid–base catalysis.

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Figure 3.10 Schematic of the secondary structures of the hairpin and VS ribozymes drawn to highlight the equivalence of the two active sites.89 The secondary structures are arranged to bring the interacting internal loops side by side, showing the A and B loops for the hairpin ribozyme (shown left) and the substrate and A730 loops for the VS ribozyme (shown right). Strand polarities in the active sites (boxed) are identical. Note that the key components including the scissile phosphate (encircled P) and the critical adenine (A) and guanine (G) nucleobases are equivalently positioned in each ribozyme. Although the overall structures of the two ribozyme have little in common, the two active sites are topologically the same.

two internal loops. In both cases, an active guanine lies on the opposing strand of the internal loop harboring the scissile phosphate and is implicated in activation of the 20 -OH nucleophile, while an active adenine is provided by the second loop and probably interacts with O50 .22,34 The topologies of the two ribozymes appear identical when the polarity of the strands is considered (Fig. 3.10), and the structures of the two active sites are very similar. So do these ribozymes share a common mechanism using general acid–base catalysis? From the outset, it was recognized that G8 and A38 of the hairpin ribozyme might participate in general acid–base catalysis.34,38,68,80 This hypothesis was expounded with great clarity by Bevilacqua,61 who showed that the observed pH dependence of the reaction was consistent with general

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acid–base catalysis by an adenine and a guanine, and that the observed rates of reaction could be achieved despite the penalty incurred in having pKa values shifted from neutrality. However, the function of G8 has been controversial, with some favoring the hypothesis that G8 performs a structural role in which it contributes to the positioning of the nucleophile and stabilizes the transition state. The origin of the controversy stems mainly from the pH dependence of the hairpin ribozyme reaction rates. The pH dependence of the ribozyme superficially appears to correspond to a single pKa for both cleavage and ligation, typically near pH 673,83 (Fig. 3.11). In principle, this is consistent with a single titratable group that could not plausibly be a guanine. If the cleavage rate responded to the ionization of a single group then the pH dependence should invert for the reverse (ligation) reaction, which is not observed.73,83 On the other hand, if the pH dependence results from the ionization of two groups (even though the upper pKa may be too high to

1

1

fA fB

fA

fA fB

fB

(pKa = 5.2)

(pKa = 8.5)

(pKa = 10)

(pKa = 6)

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fA . fB

10-6

10-6 4

5

6

7

8

9

pH

10

4

5

6

7

8

9

10

pH

Figure 3.11 Simulations of the pH dependence of substrate cleavage reactions by the VS and hairpin ribozymes. Active ribozyme requires a protonated acid (fraction fA) and an unprotonated base (fraction fB), and the reaction rate at a given pH will be proportional to the product fA  fB. These values have been calculated for two sets of values reflecting the VS ribozyme (acid pKa ¼ 5.2; base pKa ¼ 8.5; shown left) and the hairpin ribozyme (acid pKa ¼ 6; base pKa ¼ 10; shown right). The values fA (red), fB (blue), and fA  fB (black) are plotted on a logarithmic scale as a function of pH. The shaded regions indicate extremes of pH where measurement of accurate rates is difficult. Note that the simulation of the VS ribozyme cleavage rate gives a bell-shaped profile in the observable region of pH. However, the high pKa of the base for the hairpin ribozyme means that the reduction of fA  fB occurs at pH values higher than that experimentally accessible such that the cleavage rate appears to form a plateau at higher pH. The rate nevertheless reflects the ionization of two groups.

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be experimentally observable in the unmodified ribozyme), then identical profiles will be observed for both cleavage and ligation reactions. In fact, replacing G8 with nucleobases of lower pKa provides good evidence for participation of the nucleobase at this position, and thus for general acid–base catalysis by two nucleobases. Substitution of G8 by diaminopurine or 2-aminopurine,80 imidazole,85 or 8-azapurine90 resulted in cleavage becoming slower at higher pH, consistent with general acid–base catalysis involving two nucleobases. We have extensively analyzed the available evidence for the catalytic mechanism of the hairpin ribozyme and do not propose to repeat these arguments here.89 We conclude that the available data are consistent with general acid–base catalysis by A38 and G8, and that there are no data that conflict with this mechanism. Recent new data further support this.91 We have shown that the large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 50 -oxygen atom at the scissile phosphate by sulfur. This is consistent with A38 acting as general acid in the unmodified ribozyme, but would not be expected if it were only stabilizing the transition state by hydrogen bonding or electrostatic interaction. The cleavage rate by the A38P ribozyme of the 50 -PS substrate was found to increase in a log-linear manner with pH, indicative of a requirement for a deprotonated base with a relatively high pKa. Moreover, when G8 was substituted by diaminopurine, the 50 -PS substrate cleavage rate increased with pH to  6, above which the rate stayed constant. The apparent pKa was consistent with this nucleotide acting in general base catalysis. We conclude that only general acid–base catalysis by A38 and G8 is consistent with the experimental data. Despite the very different overall architectures of the hairpin and VS ribozymes, it seems that their active sites and catalytic mechanisms are likely to be quite similar with key components having the same functions (i.e., the adenine and guanine nucleobases acting in the cleavage reaction as the general acid and general base, respectively).

3. GENERAL THEMES It is clear that there is a strong mechanistic similarity between the hairpin and VS ribozymes, and that nucleobase-mediated general acid–base catalysis makes a major contribution to the rate enhancements of both ribozymes. Both employ a guanine as general base and adenine as general acid in cleavage, and the active sites of the ribozymes are very similar. This

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does not exclude additional contributions to the total observed rate enhancement. Stabilization by enhanced coordination of the transition state was observed in crystal structures of the hairpin ribozyme38 and may also contribute to the VS ribozyme since the exocyclic amines of A756 and G638 are necessary for full activity,21,22 although electrostatic effects may also contribute. However, the pH dependence of a G638DAP-substituted VS ribozyme suggested that proton transfer contributes at least 100–1000fold to the catalytic power of the ribozyme.22 General acid–base catalysis is therefore likely to be a very significant source of catalytic rate enhancement. General acid–base catalysis is not restricted to the hairpin and VS ribozymes, but seems to be the major contributor to the rate enhancement for all the nucleolytic ribozymes. In all cases, nucleobases seem to act as direct participants and guanine is the most commonly used. Guanine nucleobases appear to be strong contenders for the role of general base in cleavage reactions catalyzed by the hammerhead ribozyme (G12)92,93 and GlmS ribozyme (G33 or G40 depending on the organism).94–96 However, the guanine and adenine combination is only observed in the hairpin and VS ribozymes. The hammerhead ribozyme is proposed to employ a 20 -hydroxyl,97 and the GlmS ribozyme uses an exogenous glucosamine-6-phosphate as general acid in cleavage reactions,94,98 while the HDV ribozyme uses a Mg2þ ion to activate the nucleophile, either as general base or possibly as a Lewis acid,99,100 and a cytosine nucleobase as general acid.63,100–102 Thus it seems probable that general acid–base catalysis is a source of catalytic rate enhancement in all the nucleolytic ribozymes. By contrast, the self-splicing intron ribozymes103–106 and ribonuclease P107,108 have adopted a different strategy. They act as metalloenzymes that use bound metal ions to activate, orient, and stabilize catalytic components. As we have pointed out earlier, RNA is essentially recapitulating the two alternative strategies of protein enzymes that carry out phosphoryl transfer reactions exemplified by ribonuclease A (using two histidine side chains in general acid–base catalysis)109 and many other nucleases and polymerases that use a two-metal ion mechanism.110 As a polyelectrolyte, it is not difficult for RNA to create selective metal ion binding pockets. Thus it seems probable that the metalloenzyme mechanism is likely to have arisen before general acid–base catalysis in the chemical evolution of life, and clearly the nucleobases suffer a significant disadvantage having pKa values far from neutrality. Nevertheless, the nucleolytic ribozymes are as efficient as they need to be in their contemporary biological roles.

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ACKNOWLEDGMENTS We thank our coworkers and collaborators for many discussions on ribozyme mechanism, especially Stephanie Kath-Schorr, Shinya Harusawa, and Joe Piccirilli, and Cancer Research UK for financial support.

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41. McLeod AC, Lilley DMJ. Efficient, pH-dependent RNA ligation by the VS ribozyme in trans Biochemistry. 2004;43:1118–1125. 42. Hiley SL, Collins RA. Rapid formation of a solvent-inaccessible core in the Neurospora Varkud satellite ribozyme. EMBO J. 2001;20:5461–5469. 43. Sood VD, Yekta S, Collins RA. The contribution of 2’-hydroxyls to the cleavage activity of the Neurospora VS ribozyme. Nucleic Acids Res. 2002;30:1132–1138. 44. Ryder SP, Strobel SA. Nucleotide analog interference mapping. Methods. 1999;18:38–50. 45. Jones FD, Strobel SA. Ionization of a critical adenosine residue in the Neurospora Varkud Satellite ribozyme active site. Biochemistry. 2003;42:4265–4276. 46. Lipfert J, Ouellet J, Norman DG, Doniach S, Lilley DMJ. The complete VS ribozyme in solution studied by small-angle X-ray scattering. Structure. 2008;16:1357–1367. 47. Michiels PJA, Schouten CHJ, Hilbers CW, Heus HA. Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site. RNA. 2000;6:1821–1832. 48. Flinders J, Dieckmann T. A pH controlled conformational switch in the cleavage site of the VS ribozyme substrate RNA. J Mol Biol. 2001;308:665–679. 49. Pley HW, Flaherty KM, McKay DB. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994;372:68–74. 50. Scott WG, Finch JT, Klug A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell. 1995;81:991–1002. 51. Hoffmann B, Mitchell GT, Gendron P, et al. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site. Proc Natl Acad Sci USA. 2003;100:7003–7008. 52. Pereira MJ, Nikolova EN, Hiley SL, Jaikaran D, Collins RA, Walter NG. Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis. J Mol Biol. 2008;382:496–509. 53. Poon AH, Olive JE, McLaren M, Collins RA. Identification of separate structural features that affect rate and cation concentration dependence of self-cleavage by the Neurospora VS ribozyme. Biochemistry. 2006;45:13394–13400. 54. Ouellet J, Byrne M, Lilley DMJ. Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes. RNA. 2009;15:1822–1826. 55. Sood VD, Collins RA. Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop. J Mol Biol. 2002;320:443–454. 56. Sood VD, Beattie TL, Collins RA. Identification of phosphate groups involved in metal binding and tertiary interactions in the core of the Neurospora VS ribozyme. J Mol Biol. 1998;282:741–750. 57. Hiley SL, Sood VD, Fan J, Collins RA. 4-thio-U cross-linking identifies the active site of the VS ribozyme. EMBO J. 2002;21:4691–4698. 58. Zhao Z, McLeod A, Harusawa S, et al. Nucleobase participation in ribozyme catalysis. J Am Chem Soc. 2005;127:5026–5027. 59. Tzokov AB, Murray IA, Grasby JA. The role of magnesium ions and 2’-hydroxyl groups in the VS ribozyme-substrate interaction. J Mol Biol. 2002;324:215–226. 60. Araki L, Morita K, Yamaguchi M, et al. Synthesis of novel C4-linked C2-imidazole ribonucleoside phosphoramidite, its application to probing the catalytic mechanism of a ribozyme. J Org Chem. 2009;74:2350–2356. 61. Bevilacqua PC. Mechanistic considerations for general acid–base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry. 2003;42:2259–2265. 62. Smith MD, Collins RA. Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme. Proc Natl Acad Sci USA. 2007;104:5818–5823.

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