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A guanine nucleobase important for catalysis by the VS ribozyme

2007; Springer Nature; Volume: 26; Issue: 10 Linguagem: Inglês

10.1038/sj.emboj.7601698

1460-2075

Timothy J. Wilson, Aileen C. McLeod, David M.J. Lilley,

Advanced biosensing and bioanalysis techniques

Article26 April 2007free access A guanine nucleobase important for catalysis by the VS ribozyme Timothy J Wilson Timothy J Wilson Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author Aileen C McLeod Aileen C McLeod Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author David M J Lilley Corresponding Author David M J Lilley Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author Timothy J Wilson Timothy J Wilson Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author Aileen C McLeod Aileen C McLeod Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author David M J Lilley Corresponding Author David M J Lilley Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK Search for more papers by this author Author Information Timothy J Wilson1, Aileen C McLeod1 and David M J Lilley 1 1Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, UK *Corresponding author. Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 384243; Fax: +44 1382 385893; E-mail: [email protected] The EMBO Journal (2007)26:2489-2500https://doi.org/10.1038/sj.emboj.7601698 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid–base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid–base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution. Introduction The nucleolytic ribozymes bring about the site-specific cleavage or ligation of the backbone of RNA, with an acceleration of around a million-fold or greater. The chemical origins of catalysis are not fully understood, but significant progress has been made recently. The cleavage reaction requires the in-line attack of a 2′-O on the adjacent 3′-P with concomitant rupture of the bond to the 5′-O, in an SN2 reaction that passes through a trigonal bipyramidal phosphorane transition state. In principle, this could be catalysed by a number of processes, including substrate orientation and stabilization of the transition state. In addition, general acid–base catalysis could be important, increasing the strength of the attacking nucleophile by deprotonation and stabilization of the departing oxyanion by protonation. However, compared with proteins, the catalytic resources of RNA are rather limited, being restricted to the nucleobases and water coordinated to metal ions. There is good evidence for the direct participation of nucleobases in a number of nucleolytic ribozymes. Crystal structures of the hairpin ribozyme (Rupert and Ferré-D'Amaré, 2001; Rupert et al, 2002) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, making a number of hydrogen bonds that should stabilize the transition state. They are well placed to act in general acid–base catalysis, consistent with the pH dependence of the reaction (Bevilacqua, 2003), and its variation with functional group modifications (Pinard et al, 2001; Kuzmin et al, 2004, 2005) and replacement by imidazole (Wilson et al, 2006). The positions of the bases are consistent with a role for guanine as the general base and adenine as the general acid in the cleavage reaction. Recent crystallographic data on the extended form of the hammerhead ribozyme suggest a similar role for a guanine base (G12) (Martick and Scott, 2006), also supported by chemical data (Han and Burke, 2005). General acid–base catalysis also seems to be very important in the hepatitis delta virus ribozyme, but the participants are different. A cytosine base (C75) is juxtaposed with the scissile phosphate (Ferré-d'Amaré et al, 1998; Ke et al, 2004); substitution of this base leads to a marked loss of activity that can be partially restored by exogenous imidazole (Perrotta et al, 1999). In this ribozyme, the second participant appears to be hydrated metal ion (Nakano et al, 2000). Experiments in which the leaving group has been labilized by phosphorothiolate substitution show that the cytosine nucleobase is the general acid in the catalysis (Das and Piccirilli, 2005). Unlike the other nucleolytic ribozymes, there is no crystal structure for the VS ribozyme. Nevertheless, it has been possible to generate a model of the structure based on the known secondary structure (Beattie et al, 1995) (Figure 1), together with biophysical analysis of the component three-way helical junctions (Lafontaine et al, 2001a, 2002a). The structure is based upon the coaxial stacking of helices IV, III and VI, from which helices V and II extend laterally. This structure is in good agreement with recent studies by small-angle X-ray scattering in solution (unpublished data). The substrate stem-loop is connected through the end of helix II in the cis form of the ribozyme and makes a loop–loop interaction with helix V (Rastogi et al, 1996). These constrain both ends, and suggest that it is located in the cleft formed between helices II and VI (Lafontaine et al, 2002a). The internal loop containing A730 within helix VI has attracted particular interest as a candidate component of the active site (Lafontaine et al, 2001b; Sood and Collins, 2002). Moreover, one nucleotide in particular within the A730 loop appears to be critical for catalytic activity; any substitution of A756 leads to a loss of activity of three orders of magnitude (Lafontaine et al, 2001b; Sood and Collins, 2002), and functional group changes indicate that the Watson–Crick edge of the adenine nucleobase is especially important (Lafontaine et al, 2002b). Nucleotide analog interference mapping experiments showed that A756 is the single most sensitive nucleotide to substitution in the ribozyme (Jones and Strobel, 2003). The A730 loop may be readily juxtaposed with the substrate loop containing the scissile phosphate in the structural model of the ribozyme (Lafontaine et al, 2002a), and physical proximity was demonstrated by the observation of a UV-induced crosslink between a thiouridine placed adjacent to the scissile phosphate and A756 (Hiley et al, 2002). There is evidence consistent with a role for A756 in general acid–base chemistry. Substitution of the nucleobase of A756 with variants of altered pKA resulted in changes of ligation rate with pH, indicative of proton transfer in the transition state (Jones and Strobel, 2003), and substitution by a novel imidazole nucleoside led to a significant level of cleavage and ligation activity (Zhao et al, 2005). Figure 1.The VS ribozyme. The sequence of the ribozyme in its trans-acting form, comprising the substrate stem-loop (helix I) and the ribozyme (helices II–VI). The site of cleavage is arrowed. The A730 loop within helix VI (shaded) is the putative active site of the ribozyme. Download figure Download PowerPoint The possibility that A756 could be acting as a general acid or base raises the question of what might be acting as a partner in the transfer of protons in the transition state. It is unlikely that a hydrated metal ion plays this role, analogous to that in the HDV ribozyme, since cleavage proceeds at a significant rate in the absence of divalent metal ions (Murray et al, 1998), unlike the HDV ribozyme. We therefore considered the possibility that a second nucleobase might participate in the catalytic chemistry, analogous to the situation in the hairpin ribozyme. We can exclude most of the ribozyme, as substitutions can be tolerated so long as secondary structure is preserved, together with the lengths of helices III and V (Lafontaine et al, 2001a, 2002a). Moreover, physical access seems improbable for much of the ribozyme in terms of present models of the structure. Strongly disabling substitutions have been identified in the three-way junctions, but these correlate well with resulting changes to the folding of the ribozyme. Even the base pairs flanking the A730 loop can be reversed without great loss of activity. Substitutions of G757 lead to significant loss of cleavage and ligation activity, but remain considerably more active than A756 variants, suggesting that G757 does not play a critical role in the catalytic chemistry. This leaves one region for consideration, the internal loop of the substrate stem-loop. We have therefore examined the effect of sequence substitutions within this region. In this study, we have identified a guanine base that is critical for catalysis and is a strong candidate for the second nucleobase acting in general acid–base catalysis. Results Identification of an important nucleobase in the substrate stem-loop We have explored the importance of a number of nucleotides in the substrate stem-loop of the VS ribozyme, in the search for a catalytic nucleobase. The upper stem loop Ib is either regular duplex or involved in a loop–loop interaction with the loop of helix V in the ribozyme. We have therefore focused on the internal loop, which is believed to interact with the 730 loop of the ribozyme (Lafontaine et al, 2001b; Hiley et al, 2002). The cleavage activity under standard conditions (50 mM Tris (pH 8.0), 10 mM MgCl2, 25 mM KCl and 2 mM spermidine at 37°C) of sequence variants was examined using the trans form of the ribozyme under single-turnover conditions. (Table I). An A639G substitution had only a very small effect on cleavage rate. A621 is adjacent to the scissile phosphate, and substitution by guanine led to a 40-fold reduction in cleavage rate under standard conditions. A622U substitution led to a cleavage rate of 3.3 × 10−4 min−1. Although this is a significant loss of activity, it is likely to result from perturbation to the structure of the loop, as a uridine at position 622 could basepair with G638 in the five-nucleotide form of the loop (Andersen and Collins, 2000), extending helix Ib. Moreover, it would be difficult for a nucleobase at position 622 to interact with the scissile phosphate on the same strand. Table 1. VS ribozyme cleavage rates measured under standard conditions for different substrates Substrate Rate (min−1) Error (min−1) Relative decrease Natural 0.72 0.15 1 A621G 0.018 0.002 40 A622U 3.3 × 10−4 7 × 10−5 2200 A639G 0.42 0.06 1.7 G638A 9.9 × 10−5 9 × 10−6 7300 G638C 9 × 10−5 1 × 10−5 8400 G638U 6 × 10−5 1 × 10−5 12 000 G638 purine 9.0 × 10−5 9 × 10−6 8000 G638 diaminopurine 4.6 × 10−4 2 × 10−5 1600 G6382 aminopurine 8.5 × 10−5 7 × 10−6 8500 G638 inosine 0.026 0.004 27 G638A, C755Aa 8 × 10−5 1 × 10−5 9100 G638A, C755Ga 7 × 10−5 2 × 10−5 9800 G638A, C755Ua 9 × 10−5 3 × 10−5 8200 a The rate of cleavage of the G638A substrate was measured using variant ribozyme in which C755 was replaced by adenine, guanine or uracil. The largest effects on the rate of cleavage resulted from changes at position 638. Replacement of this guanine by any other nucleotide led to markedly impaired cleavage, with very little cleavage detectable after an hour (Figure 2). Incubation of the G638A substrate over much longer time courses revealed that the rate of cleavage is 9.9 × 10−5 min−1, reduced 104-fold compared with the natural sequence. The magnitude of this effect is even greater than that arising from an A756G substitution (Lafontaine et al, 2001b), and suggests an important role of G638 in the function of the ribozyme. Figure 2.Sequence substitutions at position 638 in the substrate strongly impair cleavage by the VS ribozyme. Products of VS ribozyme cleavage in trans on the substrate with the natural G638, and substitution by A, U or C. Cleavage reactions were performed on radioactively 5′-32P-labelled substrates under single-turnover conditions at 37°C in standard VS buffer, that is, 50 mM Tris (pH 8.0), 10 mM MgCl2, 25 mM KCl, 2 mM spermidine. The natural substrate was reacted for 5 min and the three variants for 60 min. Substrate and product were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions and visualized by phosphorimaging. Tracks: 1, G638A; 2, G638U; 3, G638C and 4, G638 natural sequence substrate. Download figure Download PowerPoint The secondary structure of the G638A substrate is not significantly altered It was important to examine the trivial possibility that the impairment of VS ribozyme cleavage of the G638A substrate results from altered folding. We compared the secondary structure of the natural and G638A substrates using the method of in-line probing (Soukup and Breaker, 1999). In this approach, radioactively 5′-32P-labelled RNA is subjected to a prolonged incubation in the presence of buffer and Mg2+ ions. Where the backbone is relatively flexible, the RNA can locally sample a conformation in which the 2′-OH may carry out an in-line nucleophilic attack on the 3′-phosphate, but this is hindered in more rigid parts of the molecule, including duplex regions. We performed the analysis under conditions similar to those used for most of the ribozyme cleavage experiments (Figure 3). The helical regions of both species were clearly protected from cleavage, whereas the terminal loop and the formally single-stranded regions at the lower end of the helix were subject to significant cleavage. Evidently the general secondary structure of the substrate is unaltered by the G638A substitution. Cleavage immediately adjacent to position 638 is enhanced in the G638A substrate relative to the natural sequence. This is a very local effect, and increased sensitivity at this position is also observed in hydroxide cleavage of the same RNA under fully denaturing conditions. But the in-line probing indicates that no gross conformational change results from the substitution. Figure 3.Comparison of the conformations of the natural and G638A substrates by in-line probing. Versions of natural sequence and G638A substrates with 5′ and 3′ terminal extensions of deoxyribonucleotides were synthesized to improve the electrophoretic resolution of the RNA sections. Radioactively 5′-32P-labelled substrates were incubated in standard VS buffer at 25°C for 40 h. Cleavages were analysed by electrophoresis in a 20% polyacrylamide gel under denaturing conditions. Tracks 1 and 4, base cleavage of natural and G638A substrates, respectively and tracks 2 and 3, in-line probing analysis of natural and G638A substrates, respectively. The scheme shows the sequence of the natural substrate, with the arrow indicating the position of ribozyme cleavage. The positions of sensitivity to in-line probing are indicated by filled circles, the size of which reflects the extent of cleavage. The open circle shows the position of the phosphodiester linkage that is more sensitive in the G638A substrate; note that this position also exhibits enhanced base cleavage. The shorter fragments migrate as doublets, due to resolution of the cyclic 2′3′-phosphates and their products of hydrolysis during the long incubation. Download figure Download PowerPoint A G638A substrate exhibits unperturbed binding to the VS ribozyme Another possible explanation for the low rate of cleavage of a G638A substrate by the VS ribozyme is impaired binding. If the G638A substrate is able to bind to the ribozyme in a manner that is similar to that of the normal substrate, it is likely to act as a competitive inhibitor, described by the scheme shown in Figure 4A. We therefore carried out cleavage reactions under standard conditions, in the presence of different concentrations of G638A substrate. The progress curves (Figure 4B) show a marked reduction in cleavage rate with increased concentration of G638A substrate. The observed rates, kobs, have been fitted to the equation for competitive inhibition that is, Figure 4.Affinity and rate of substrate binding in trans to the VS ribozyme. (A) Reaction scheme for the ribozyme cleavage in the presence of the variant substrate. The natural sequence substrate (SG) binds to the ribozyme (Rz) with an affinity KS to form a non-covalent complex that undergoes the cleavage reaction at rate k2. The G638A substrate binds to the ribozyme with an affinity KI. The variant substrate undergoes a negligible amount of cleavage during the incubation, and therefore simply acts as an inhibitor of the reaction. (B) Cleavage of a natural sequence substrate by VS ribozyme was performed in trans under standard single-turnover conditions in the presence of different concentrations of G638A substrate. Progress curves are shown for reactions carried out in the presence of the following concentrations of G638A substrate: 0 (filled circles), 0.3 (open circles), 0.8 (filled squares), 1.4 (open squares), 2.5 (filled diamonds) and 5 μM (open diamonds). In the inset, the observed rate constants (kobs) are plotted as a function of G638A substrate concentration and fitted to equation 1, appropriate for competitive inhibition by the variant substrate. (C) Substrate cleavage by VS ribozyme that had been preincubated with G638A variant substrate. Ribozyme (1 μM, with or without 2.8 μM G638A substrate) and substrate were separately incubated followed by mixing together at 0 time. The progress of both reactions is shown: no G638A substrate (open circles); plus G638A substrate (filled circles) and fitted to single exponentials, yielding rates of 0.77±0.03 and 0.28±0.01 min−1. The data for the first 0.6 min are shown expanded in the inset. Note that no lag phase is discernible. (D) A VS cleavage reaction interrupted by addition of G638A substrate. A cleavage reaction was initiated by addition of 10 μl of 1 μM ribozyme to 10 μl of natural sequence substrate, followed by addition of 10 μl of 30 μM G638A substrate, 1 μM ribozyme after 60 s. Progress curves are plotted for the interrupted reaction (filled circles) and one reaction allowed to continue normally (open circles; these data are taken from panel C), and fitted to single exponentials. There is no discernible intermediate phase, and the curves intersect at 64.8 s. Download figure Download PowerPoint where [Rz] is the ribozyme concentration, [SA] is the concentration of the G638A substrate (treated as an inhibitor in this analysis), k2 is the rate of conversion of substrate to product complex, KM is the Michaelis constant for the substrate and KI is the dissociation constant for the G638A substrate. Values of KM=0.54 μM and KMapp=1 μM have been measured by multiple- (Tzokov et al, 2002) and single-turnover (Lafontaine et al, 2001b) experiments, respectively, and for the present analysis, we have assumed that KM=1 μM. The data are well fitted by this equation, consistent with the G638A substrate acting as a competitive inhibitor of the cleavage of the natural substrate, and yielding a value of KI=0.6±0.1 μM. Thus, the binding affinities of the natural and G638A substrates are closely similar. To investigate the rates of substrate binding, we compared the rate of cleavage of the natural substrate by 1 μM VS ribozyme under standard conditions and by 1 μM VS ribozyme that had been preincubated with 2.8 μM G638A substrate (Figure 4C). The progress curves show the expected reduction in cleavage rate in the presence of the G638A substrate, while the absence of a lag in the early part of the time course indicates that dissociation of the G638A substrate is either very fast or very slow. To distinguish between these possibilities, the progress of the cleavage reaction of the natural substrate was interrupted by the addition of an excess of G638A substrate after 60 s (Figure 4D). The adjustment of cleavage rate occurs within 5 s, showing that the dissociation of the natural substrate and the association of the G638A substrate are rapid. These experiments demonstrate that the rate-limiting step for cleavage must be subsequent to binding. Potential basepairing by G638 A further possibility is that G638 is required to basepair with a cytosine to allow the substrate and A730 loops to interact productively, analogous perhaps to the basepairing between G+1 and C25 in the hairpin ribozyme (Rupert and Ferré-D'Amaré, 2001). Were this to be the case, it would be necessary to identify the participating cytosine. We have previously shown that the base pairs flanking the A730 loop can be reversed with relatively low penalties to cleavage rate (Lafontaine et al, 2001b), so these are unlikely to be involved. This leaves C755 within the A730 loop as the only candidate nucleotide. Cleavage is only weakly affected by any change other than C755G (Lafontaine et al, 2001b), but a C755A variant is significantly impaired in ligation (McLeod and Lilley, 2004). We examined the rates of cleavage of the G638A substrate by VS ribozymes with C755 replaced by either A, G or U. In all cases, extremely slow rates of cleavage were obtained (Table I), with no evidence of restoration of activity when an A–U basepair might replace one between G638 and C755. Importance of functional groups at position 638 The data presented above demonstrate that G638 plays a direct role in the central conversion of substrate to product. We therefore explored the importance of different functional groups at position 638. It has been shown that the 2′-OH is not important in the cleavage reaction (Tzokov et al, 2002), and we therefore focused on the nucleobase (Figure 5). Cleavage rates are collected in Table I. Removal of the exocyclic carbonyl and amine groups (purine) resulted in a similarly low rate of cleavage as G638A, as did removal of the C6 carbonyl group alone (2-aminopurine). Replacement of the C6 carbonyl group by an amine (2,6-diaminopurine) also led to very impaired cleavage. By contrast, removal of the C2 amine with retention of the carbonyl (hypoxanthine, that is, an inosine nucleoside) resulted in a significant rate of cleavage, 27-fold slower than the natural sequence. The results suggest that the main requirement for the nucleobase at position 638 for cleavage under standard conditions is the C6 carbonyl, or the imino proton on N1, or both. Figure 5.Effect of functional group changes at position 638 on the rate of VS ribozyme cleavage. Variant substrates were synthesized with alternative nucleotide bases or analogs at position 638 as shown and radioactively 5′-32P labelled. These were subjected to cleavage in trans by VS ribozyme under single-turnover conditions for 30 min at 37°C in standard buffer. Substrate and product were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions, and visualized by phosphorimaging. Tracks 1, natural substrate before cleavage; tracks 2–8, incubation with VS ribozyme; track 2, natural substrate; track 3, substitution by adenine; track 4, substitution by inosine; track 5, substitution by 2-aminopurine; track 6, substitution by 2,6-diaminopurine and track 7, substitution by purine. Download figure Download PowerPoint The pH dependence of cleavage rates Drawing analogies with other nucleolytic ribozymes, G638 may participate in general acid–base catalysis of the transesterification reactions, probably in concert with A756 in the light of previous data. If so, the ribozyme is required to be in the correct state of protonation to be active, and this could be reflected by the pH dependence of the observed rate of cleavage. We were concerned that a dependence of reaction rate upon divalent ion concentration might distort the measured pH dependence, and therefore made an initial study of the dependence of cleavage upon Mg2+ concentration. We found that the rate of cleavage was near-maximal in the presence of 200 mM Mg2+ at both pH 5.5 and 8 (data not shown). We therefore performed all our studies of pH dependence in the presence of in 200 mM MgCl2, 25 mM KCl and 50 mM buffer at the required pH; under these conditions, the natural sequence substrate was cleaved at a rate of ∼5 min−1 at pH 8. We have found it helpful to simulate the pH dependence expected for the participation of nucleobases of different pKA to aid the interpretation of experimental data. Figure 6A shows the protonated and deprotonated fractions of an acid (fraction fA) of pKA 5.5 and base (fB) of pKA 9.0, which might correspond to an adenine base (with its pKA raised by the electronegative environment) and guanine base respectively. The product fA·fB is then the fraction of ribozyme in the correct form to perform catalysis. The observed rate of cleavage should follow the pH dependence determined by fA·fB. The shaded regions correspond to the extreme pH values inaccessible to experimental study. The rate of reaction rises at lower pH, reaching a maximum around neutrality due to deprotonation of the acid. At increased pH, the rate declines again, due to almost complete deprotonation of the base. This may or may not be within the accessible region; in the hairpin ribozyme, this is barely observable (Kuzmin et al, 2004; Wilson et al, 2006). The shape of the fA·fB curve is unaltered if the acid and base pKA values are exchanged (Bevilacqua, 2003). Figure 6.Calculated pH dependence of the cleavage reaction of the VS ribozyme as a function of base pKA, assuming general acid–base catalysis. The fractions of protonated acid fA, unprotonated base fB and their product fA·fB have been calculated and plotted as a function of pH, following the approach of Bevilacqua (2003). The shaded sections are the regions of pH not accessible to experimental study. Reaction rate should be proportional to the fraction of ribozyme in the appropriate state of protonation, that is, fA·fB. Note that in these graphs, fA and fB are plotted on a log10 scale (left), whereas fA·fB is plotted on a linear scale (right). (A) Plot for pKA values of 5.5 and 9 for the acid and base, respectively. This might correspond to the natural ribozyme, assuming that the acid is an adenine with an elevated pKA and the base is a guanine with a slightly reduced pKA. The predicted reaction rate profile is a broad bell shape, with a maximum close to neutrality. (B) Plot for pKA values of 5.5 and 5 for the acid and base, respectively. This situation could emerge if the guanine were replaced by a nucleobase of significantly lower pKA (e.g. adenine or DAP). The reaction rate is predicted to exhibit a marked increase at low pH, with a maximum that is just detectable for these values. Download figure Download PowerPoint The pH dependence of the cleavage rate of the natural substrate (i.e. with guanine at position 638) is shown in Figure 7A. An increase of rate with pH is clearly observable up to pH 7, and declines with further increase in pH. The data have been fitted to a double-ionization model (Bevilacqua, 2003), giving apparent pKA values of 5.2±0.1 and 8.4±0.1. While other explanations are possible (see Discussion), this pH profile is consistent with the hypothesis that A756 and G638 participate in acid–base catalysis. The lower pKA is significantly higher than free adenosine mononucleotide (3.7; Dawson et al, 1986), but corresponds well to values measured previously by NMR for adenine bases in catalytic RNA molecules (Legault and Pardi, 1997; Ravindranathan et al, 2000). A value of 5.6 was found for the trans ligation reaction of the VS ribozyme under standard buffer conditions (McLeod and Lilley, 2004). That study, and an earlier one of the cleavage reaction (Guo and Collins, 1995), did not show a significant decrease in rate at high pH, consistent with an upper pKA lying outside the observable range; the pKA of free GMP is 9.4 (Dawson et al, 1986). The lower apparent pKA values observed under present conditions could be a consequence of the 20-fold higher concentration of Mg2+ ions used in this study. Figure 7.pH dependence of observed rates for VS ribozyme cleavage reactions in trans with the natural and modified substrates. Cleavage reaction rates were measured under single-turnover conditions in 50 mM MES, HEPES or TAPS of required pH containing 200 mM MgCl2 and 25 mM KCl. All rates are averages of ⩾3 measurements, and the error bars indicate one standard deviation. All data have been fitted to a double-ionization model in which there is a requirement for one protonated and one deprotonated form (equation 2), from which apparent pKA values have been determined. (A) The pH dependence of the rate of cleavage of the natural sequence substrate. The data are well fitted