Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Abstract

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK_a = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756.

Ribozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).

Fig. 1.

The sequence of the VS ribozyme, the proposed mechanism, and the chemical structure of the 5′-phosphorothiolate substitution. (A). The sequence of the trans-acting ribozyme and substrate used here. The cleaved bond is arrowed. (B). Mechanism of general acid-base catalysis for the cleavage reaction of the VS ribozyme. (C). The chemical structure of a protected GpA dinucleotide with a 5′-phosphorothiolate linkage. oNO₂Bn = o-nitrobenzyl. (D). The chemical structure of 2,6-diaminopurine nucleoside.

The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5–8).

In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10–13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model places G638 and A756 in proximity to the scissile phosphate (14).

At high concentrations of Mg²⁺ ions, the pH dependence of the cleavage reaction of the trans-acting VS ribozyme is bell shaped, fitting a model involving proton transfers in the transition state with participating groups of pK_a = 5.2 and 8.4 (9). Substitution of G638 by diaminopurine shifts the pH profile, corresponding to new pK_a values of 4.6 and 5.6. A plausible mechanism involves general acid-base catalysis by A756 and G638, but it is not possible to determine from the pH dependence which nucleobase acts as the acid, and which the base. The alternatives predict identical pH profiles. Smith et al. have suggested a resolution of this ambiguity, based on the relationship between ionic environment and nucleobase pK_a (15), but there is no direct evidence enabling the assignment of function to specific nucleobases.

A similar ambiguity existed for the HDV ribozyme, where the critical functionalities are a cytosine nucleobase (16–18) and a metal ion-bound water (2). Assignment of the general acid and base was both difficult and controversial. The distinction was made by the introduction of a 5′-phosphorothiolate (5′-PS) substitution at the scissile phosphate (19). The 5′ sulfur atom is a much better leaving group than oxygen and therefore no longer requires protonation by a general acid. Thus alterations to the ribozyme that impair the function of the general acid (so inhibiting cleavage of the oxy substrate) should have little effect on cleavage of a 5′-PS-containing substrate. Moreover, the pH dependence of the cleavage rate of the 5′-PS substrate should reflect the deprotonation of the base alone. We have therefore synthesized VS ribozyme substrates containing a 5′-PS linkage at the cleavage site. The substitution restores full activity to the highly impaired A756G ribozyme, and we conclude that the nucleobase of A756 is the probable general acid in the cleavage reaction.

Results

Kinetic Analysis of Cleavage Using Modified VS Ribozyme and Substrate.

The analysis of VS ribozyme cleavage reactions was performed using a ribozyme comprising helices II to VI acting in trans upon a substrate stem loop (Fig. 1A). The ribozyme was prepared by transcription from a DNA template, and the substrate by a combination of chemical synthesis and sequential ligations (Figs. S1 and S2). A 5′-PS-substituted scissile phosphate was introduced by synthesis of a GpA dinucleotide containing a 5′ sulfur on the adenosine with o-nitrobenzyl protection on the 2′-hydroxyl of the guanosine to prevent premature activation of the nucleophile (Fig. 1C).

The cleavage of radioactively [5^′-³²P]-labeled substrate was studied under single-turnover conditions immediately following deprotection of the 2′-hydroxyl nucleophile using ultraviolet irradiation. Products of ribozyme cleavage were separated by gel electrophoresis and quantified by phosphorimaging.

5′-PS Substitution Restores Cleavage by a Ribozyme with a A756G Substitution.

We have adopted standard conditions of 50 mM MES (pH 6.0), 25 mM KCl, and 200 mM MgCl₂ at 37 °C. All the measured reaction rates are tabulated in Table 1.

Table 1.

Observed rates of substrate cleavage (k_obs) in trans measured under standard conditions

Rz	Substrate	5′-PO, kobs/ min-1	5′-PS, kobs/ min-1	kobs5′-PS/kobs5′-PO
—	wt	< 10-5	0.0012 ± 0.0001	> 120
wt	wt	2.4 ± 0.3	0.37 ± 0.03	0.16
A756G	wt	0.00023 ± 0.00006	0.84 ± 0.05	3,700
C755G	wt	0.14 ± 0.01	0.35 ± 0.03	2.5
G757A	wt	0.39 ± 0.07	0.22 ± 0.04	0.56
A730U	wt	0.0042 ± 0.0004	0.038 ± 0.002	9.0
A756C	wt	0.029 ± 0.006	0.080 ± 0.009	2.8
wt	DAP	0.037 ± 0.004	0.30 ± 0.02	8.1
A756G	DAP	0.0003 ± 0.0001	1.05 ± 0.02	3,500

Single-turnover rates were determined for the indicated combinations of ribozyme and substrate and either 5′-PO or 5′-PS substitution. Unmodified ribozyme or substrate sequence is indicated by wt. Each rate is the average of ≥3 independent measurements

Under standard conditions, in the absence of ribozyme the normal substrate carrying an 5′-oxy scissile phosphate group at A621 (5′-PO) exhibited no detectable cleavage after 15-min incubation, whereas a small fraction of product was observed for the 5′-PS substrate (Fig. 2A). This cleavage occurs selectively at the position cleaved by the VS ribozyme and is due to cleavage of the labile P-S bond during deprotection. Addition of the natural-sequence (A756) ribozyme resulted in significant cleavage of both 5′-PO and 5′-PS substrates. The 5′-PO substrate is poorly cleaved by a ribozyme carrying the A756G substitution, consistent with previous observations (10). By contrast, the 5′-PS substrate was substantially cleaved by the A756G ribozyme. The extent of product formation for this reaction is shown in Fig. 2B, and the reaction progress is plotted in Fig. 2C for each ribozyme reaction. The A756 ribozyme cleaves the 5′-PO and 5′-PS substrates at 2.4 min^-1 and 0.37 min^-1, respectively. The A756G ribozyme cleaves the 5′-PO substrate at 0.00023 min^-1, yet cleaves the 5′-PS substrate 3,700-fold faster at 0.84 min^-1. This rate of 5′-PS substrate cleavage is twice that observed for the A756 ribozyme and represents complete restoration of A756G ribozyme activity by the 5′-PS linkage. This restoration is consistent with the A756 nucleobase acting as the general acid in the unmodified cleavage reaction.

Fig. 2.

Cleavage of natural-sequence substrate by A756 or A756G VS ribozyme as a function of the presence or absence of a 5′-phosphorothiolate linkage at the scissile phosphate. (A). Gel electrophoresis of reaction products. The 5′-PO (tracks 1, 3, 5) and 5′-PS (tracks 2, 4, 6) substrates were incubated with no ribozyme (tracks 1, 2), A756 ribozyme (tracks 3, 4), or A756G ribozyme (tracks 5, 6) for 15 min. (B). Cleavage of the 5′-PS substrate catalyzed by the A756G ribozyme as a function to time (indicated above gel). (C). Plots of reaction progress with time, fitted to single exponential functions. Filled circles, 5^′-PO + A756 ribozyme; open circles, 5^′-PS + A756 ribozyme; open squares, 5^′-PS + A756G ribozyme; filled squares (Inset), 5^′-PO + A756G ribozyme. Corresponding axes use the same units.

We examined the effect of the 5′-PS modification on other variations within the A730 loop of the ribozyme. The C755G ribozyme cleaves the 5′-PO substrate at 0.14 min^-1 (17-fold slower than the natural ribozyme); this rate increased slightly to 0.35 min^-1 for the 5′-PS substrate. The G757A ribozyme cleaves the 5′-PO substrate at 0.39 min^-1 under these conditions. This was barely affected by the 5′-PS modification (a rate of 0.22 min^-1). Thus there is no evidence for a direct role for either C755 or G757 in leaving group stabilization. The A730U ribozyme is much less active, cleaving the 5′-PO substrate at a rate of 0.0042 min^-1. It cleaves the 5′-PS substrate only 9-fold faster at 0.038 min^-1. We examined a second sequence variant at the critical 756 position, A756C. This ribozyme cleaved the 5′-PO substrate at a rate of 0.0029 min^-1, 80-fold slower than the A756 ribozyme. Interestingly, it cleaved the 5′-PS substrate only 3-fold faster at a rate of 0.080 min^-1. These results suggest that cytosine can replace adenine to some degree under these conditions, unsurprisingly because the bases share an amidine functionality with similar pK_as, but the substitution results in a structure with a reduced intrinsic activity; this may reflect a change in the active site structure due to the substitution of a pyrimidine for a purine nucleobase.

If A756 is the general acid, G638 is the likely general base and inhibitory substitutions at this position are not expected to be rescued by the 5′-PS substitution. Replacement of G638 by diaminopurine (DAP, Fig. 1D) leads to significantly lower rates of substrate cleavage (9). Under standard conditions a 5′-PO substrate carrying the G638DAP substitution was cleaved by the A756 ribozyme at a rate of 0.037 min^-1. Introduction of the 5′-PS substitution to the G638DAP substrate resulted in 8-fold faster cleavage by the A756 ribozyme at a rate of 0.30 min^-1 (Table 1). While this increase is consistent with a mechanism in which G638 acts as a general base (see Discussion), it is necessary to examine the reactivity of 5′-PS substrates over a broad pH range to clarify the respective roles of G638 and A756, because the pH dependence of the cleavage rate should reflect the pK_a of the general base alone.

The Rate of Cleavage Reaction of a 5′-PS-Substituted Substrate Is Not Reduced at pH Values at Which Adenine Would Deprotonate.

The pH dependence of the cleavage rate for the natural VS ribozyme reaction is bell shaped, corresponding to the ionization of bases with apparent pK_a values of 5.2 and 8.4, assigned to A756 and G638, respectively (9). In both cases the local environment shifts the pK_a from the solution values of 3.8 for free adenosine and 9.4 for free guanosine. The pH profile changes when a G638DAP substrate is cleaved by the natural-sequence ribozyme, with apparent pK_a values of 4.6 and 5.6 assigned to A756 and DAP638, respectively, because free DAP has the higher unperturbed pK_a of 5.1 (9). We may calculate the fraction of the acid in the protonated form (f_A) and base in its deprotonated form (f_B) from the observed pK_a values. The rate of a reaction requiring both acid and base should follow the shape of the product f_A·f_B, showing the origin of the bell-shaped profile (Fig. 3A). The rate is reduced at low pH due to increased protonation of the base, and at high pH due to deprotonation of the acid. However, if labilization of the leaving group leads to a reaction that is independent of general acid catalysis, the reaction rate should depend solely on the fraction of unprotonated base, i.e., f_B (also plotted in Fig. 3A). In this case the reaction rate increases to pH ∼ 6.5 and then forms a plateau. The rate does not diminish at higher pH, because the protonated form of the acid is not required for the catalytic activity of the ribozyme.

Fig. 3.

The pH dependence of cleavage rates. (A) Simulation of reaction rate as a function of pH. (Upper) Fraction of protonated nucleobase (f_A) with pK_a = 4.6, and unprotonated nucleobase (f_B) with pK_a = 5.6 (unbroken line) (corresponding to the apparent pK_a values for the combination of A756 with DAP638), and unprotonated G638 (f_B) with pK_a = 8.4 (broken line) (9). (Lower) Comparison of f_A·f_B corresponding to a reaction catalyzed by general acid-base catalysis (pK_a = 4.6 and 5.6), with single f_B values of pK_a 5.6 and 8.4 corresponding to reactions catalyzed by general base catalysis alone using DAP and G, respectively. The shaded regions reflect pH values outside the experimentally accessible range. (B) Experimental profiles of cleavage rate as a function of pH for the G638DAP substrate with the natural-sequence ribozyme. (Upper) Rate of cleavage of the G638DAP, 5′-PS substrate plotted on a linear scale. (Lower) Logarithmic scale of cleavage rate for the G638DAP, 5′-PO (filled circles, taken from ref. 9) and G638DAP, 5′-PS (open circles) substrates. The G638DAP, 5′-PO substrate has been fitted to a double-ionization model. By contrast, the pH dependence of the G638DAP, 5′-PS substrate indicates a single ionization, consistent with a reaction not requiring general acid catalysis. (C) Experimental profiles of cleavage rate as a function of pH for the G638 5′-PS substrate with the A756 (open circles) and A756G (closed circles) ribozymes. Both give log-linear pH dependence over the range 5–6.5 with near unit gradient. The single-ionization fit for the cleavage of the G638DAP, 5′-PS substrate cleaved by natural-sequence ribozyme has been superimposed on the plot (broken line, taken from B).

We have measured the rate of cleavage of the G638DAP substrate with a 5′-PS substitution by the A756 ribozyme as a function of pH (Fig. 3B) and compared this with the profile for the 5′-PO substrate measured previously. The latter follows a bell-shaped rate profile (9), but the rate of cleavage of the 5′-PS substrate increases up to pH ∼ 6 after which the reaction rate becomes independent of pH. The data for the 5′-PS substrate fit well to a model involving a single ionization, with an apparent pK_a of 5.3. They are fully consistent with a reaction in which the cleavage of the labilized substrate is subject to general base catalysis by the diaminopurine at position 638. However, these data alone do not exclude the possibility that A756 acts as the general base, as the apparent pK_a is only 0.7 units higher than that assigned to A756 in the presence of G638DAP.

If the nucleobase at position 638 acts as the general base, then the pH dependence of the natural-sequence substrate (i.e., with G638) bearing the 5′-PS modification should correspond to a pK_a close to 8.4 (9). The expected pH dependence is simulated in Fig. 3A (broken lines). The reaction rate should follow the extent of deprotonation of the guanine, and this increases log-linearly up to approximately pH 8. Conversely, if A756 acts as the general base, the pH dependence of the natural-sequence 5′-PS substrate should follow the deprotonation of the adenine and would be similar to that of the G638DAP substrate presented in Fig. 3B. Over the observable pH range the experimental rates for the 5′-PS substrate increase log-linearly up to pH 6.5 (Fig. 3C, open circles). Thus the general base must have a pK_a greater than 7, excluding A756 from this role. The simplest, fully consistent model describing the data is that A756 acts as the general acid and G638 as the general base.

Further support for this conclusion comes from the pH dependence of cleavage of the natural-sequence 5′-PS substrate by the A756G ribozyme (Fig. 3C, closed circles). This too increases log-linearly over the observable pH range, excluding a general base with a pK_a below neutrality. We have also studied the cleavage of a G638DAP substrate by an A756G ribozyme. The A756G ribozyme cleaves the 5′-PO substrate at 0.0003 min^-1 at pH 6, similar to the cleavage rate for the natural sequence substrate. The 5′-PS substrate is cleaved at 1.05 min^-1, slightly faster than the cleavage rate of the natural-sequence substrate. Thus, even in the presence of the G638DAP substitution, activity is fully restored in the presence of the highly deleterious A756G modification.

Discussion

Significant data implicate A756 and G638 in the catalytic mechanism of the VS ribozyme (9–13). The effect of substitution at these positions and the pH dependence of rates are consistent with general acid-base catalysis by these two nucleobases (9). The change in rate in D₂O also suggests that proton transfer occurs in the rate-limiting step of cleavage (20). A756G substitution lowers cleavage activity by 4 orders of magnitude at pH 6.0, but substitution of the 5′-O leaving group by sulfur enhances the rate 3,800-fold by the A756G ribozyme. Thus, labilization of the leaving group so that protonation is no longer required to facilitate bond breakage suppresses the inhibitory effect of the A756G substitution. The A756G ribozyme cleaves the natural-sequence 5′-PS substrate twice as fast as the A756 ribozyme, suggesting that the structure of the ribozyme is not altered by the substitution so as to affect its activity. Other substitutions in the A730 loop of helix VI have smaller effects on cleavage rates than those at 756 and show similar rates for 5′-PO and 5′-PS substrates. This confirms the importance of A756 to the reaction mechanism.

Identifying A756 as the general acid in the cleavage reaction leaves G638 as the probable general base. The base would be expected to facilitate proton transfer from the 2′-O nucleophile and should still be required irrespective of the lability of the leaving group. The log-linear pH dependence below pH 7 of the cleavage of the natural-sequence 5′-PS substrate is fully consistent with general base catalysis by guanine and excludes adenine from this role. The pH dependence of the ribozyme-mediated cleavage changes markedly for the 5′-PS G638DAP substrate. The rate rises with pH and then remains constant beyond pH 6 giving a calculated pK_a of 5.3. This change in pH-rate profile induced by the G638DAP substitution strongly suggests that G638 is the general base.

The proposed roles for A756 and G638 also account for other observations. At pH 6.0 the A756 ribozyme cleaves the 5′-PS G638DAP substrate 8-fold faster than the corresponding 5′-PO substrate. The faster rate probably arises from the increase in the fraction of ribozyme in the active state. At pH 6.0, for cleavage of the 5′-PO substrate the proportion of acid and base in the active state is ∼0.03 (f_A·f_B in Fig. 3A). The active fraction is predicted to increase 20-fold for cleavage of the 5′-PS substrate where the fraction of protonated acid is not important [f_B(pK_a = 5.6) in Fig. 3A], potentially accounting for the observed increase in rate. In contrast, for the A756G ribozyme the active fraction will be the same for 5′-PO and 5′-PS substrates because guanine is fully protonated at pH 6 and thus f_A is ∼1. The low activity of the A756G ribozyme for 5′-PO cleavage is likely due to guanine being a much weaker acid than adenine. These results are therefore consistent with A756 acting as the acid and G638 as the base for the cleavage reaction (Fig. 4). By microscopic reversibility, in the ligation reaction A756 and G638 should act as base (deprotonating the 5′-O nucleophile) and acid (protonating the 2′-O leaving group), respectively.

Fig. 4.

The probable chemical mechanism of cleavage reaction of the VS ribozyme.

By itself the reduction in the rate of cleavage of the unmodified substrate by A756G ribozyme could be consistent with a number of roles for A756. The nucleobase could stabilize the reaction transition state by specific hydrogen bonding, or a protonated adenine might stabilize the dianionic phosphorane electrostatically. Both effects have been proposed for the hairpin ribozyme (3, 21). Were this to be the case in the VS ribozyme, then substitution of the nucleobase at position 756 could lead to lower cleavage rates, as observed. But these impairments should not be suppressed by activation of the leaving group by 5′-PS substitution. Our new results strongly suggest that general acid catalysis by A756 is a major contributor to the catalytic rate enhancement of the VS ribozyme.

While general acid catalysis by the A756 nucleobase is not required for the cleavage of the 5′-PS substrate, the ribozyme still contributes to catalysis of 5′-PS cleavage. Although the 5′-PS substrate undergoes cleavage at a significant rate in the absence of ribozyme (0.0012 min^-1), addition of the A756 ribozyme increases the cleavage rate 300-fold to 0.37 min^-1. It is therefore likely that the interaction of the substrate with the ribozyme leads to a structural remodeling that is required for full activity. In an NMR structure of the substrate RNA alone (22), N1 of G638 is 6 Å from the 2′-OH group of G620. Moreover, the 2′-O nucleophile is far from an in-line trajectory. It is likely that an association of the substrate loop with the A730 loop of the ribozyme would generate the structure required for catalytic activity. A similar situation exists in the hairpin ribozyme, where the active geometry results from a close interaction between the A and B loops (23). The local structure (3, 24) is significantly altered from those of the loops in isolation (25, 26). We have previously noted that the topological placement of the key components within the two interacting loops of the hairpin and VS ribozymes is very similar (9).

In summary, the VS ribozyme catalyzes the cleavage of its substrate by making an intimate loop-loop interaction with the A730 loop in helix VI. This most probably brings about a structural rearrangement that aligns the 2′-O for nucleophilic attack and moves the nucleobase of G638 into position to act as a general base to deprotonate the hydroxyl of G620 thereby enhancing its nucleophilicity. The association of the loops is expected to position A756 to protonate the 5′-oxyanion leaving group. Our results indicate that general acid catalysis contributes substantially to the observed rate enhancement by the VS ribozyme. It is very probable that the hairpin ribozyme uses an essentially identical catalytic mechanism, in which G8 and A38 are the general base and acid, respectively.

We may ask how the rate of chemical catalysis by the VS ribozyme compares with protein enzymes ? Pancreatic ribonuclease carries out the same cleavage of a phosphodiester linkage in RNA using two histidine side chains as general acid and base (27, 28), with an observed rate of ≤ 1,400 s^-1 (29). Given the pK_a for acid and base of 6.2 and 5.8, respectively (30), the maximum k_cat is ∼3,700 s^-1. The fastest observed rate of cleavage for the VS ribozyme acting in trans is ∼12 min^-1. However, the observed rate (k_obs) will be constrained by the small fraction of ribozyme molecules that have a protonated A756 (i.e., f_A) and a deprotonated G638 (f_B), i.e.,

[1]

where k_cat is the intrinsic rate of catalysis. We calculate f_A·f_B from the measured pK_a values for A756 and G638 (9) to be 3.8 × 10^-4, and hence k_cat is ∼520 s^-1. This will be even faster for the rapidly cleaving cis ribozyme (31). Similarly, the k_cat for cleavage by the HDV ribozyme has been estimated to be in the range of 10² to 10⁴ s^-1 (2). Thus, the rate of cleavage by ribozyme molecules that are in the correct ionization state appears comparable to that of RNaseA. All the nucleolytic ribozymes seem to employ general acid-base catalysis. In addition to the nucleobases, this class of ribozyme has evolved the use of bound hydrated metal ions (2) and glucosamine-6-phosphate (32, 33). All these alternatives have a pK_a far from neutrality. In general these ribozymes are required to turn over only a single time in their natural function, but the limitation of a low active fraction imposed by the unfavorable pK_a values may have been a significant factor holding back the evolution of RNA-based catalysts. Ultimately the selection of histidine with its imidazole side chain whose pK_a is close to neutrality enabled protein enzymes to achieve significantly greater rates where turnover is important.

Materials and Methods

Preparation of RNA.

VS ribozyme was transcribed from a DNA template and purified by standard methods. To prepare substrates, GpA dinucleotides were synthesized containing either a 5′ oxygen or sulfur on the adenosine and o-nitrobenzyl protection on the 2′-hydroxyl of the guanosine. Dinucleotides were first ligated to the 5′ portion of the substrate with RNA ligase I, then to the 3′ sequence using RNA ligase II and a DNA splint. The methods are fully described in SI Text.

Analysis of Ribozyme Kinetics.

The cleavage of radioactively [5^′-³²P]-labeled substrate was studied under single-turnover conditions (9). Immediately prior to use substrates were irradiated for 15 min at 365 nm to activate the 2′-hydroxyl. Following equilibration to 37 °C in reaction buffer, reactions were initiated by mixing ribozyme and substrate. Products of ribozyme cleavage were separated by gel electrophoresis and quantified by phosphorimaging. Reaction progress curves were fitted by nonlinear regression analysis to single or double exponential functions. The pH dependence of observed cleavage rates of 5′-PO substrates were fitted to a double-ionization model appropriate for analysis of general acid-base catalysis, assuming a requirement for one protonated and one deprotonated form. The pH dependence of observed cleavage rates of 5′-PS substrates were fitted to a single-ionization model in which the deprotonated form is assumed to be active. Full descriptions of kinetic methods and analysis are given in SI Text.