Nature Chemical Biology 1, 45-52 (2005)

Nature Chemical Biology 1, 45-52 (2005) doi: 10.1038/nchembio703 General acid catalysis by the hepatitis delta virus ribozyme

Subha R Das1 and Joseph A Piccirilli1

Recent crystallographic and functional analyses of RNA enzymes have raised the possibility that the purine and pyrimidine nucleobases may function as general acid-base catalysts. However, this mode of nucleobase-mediated catalysis has been difficult to establish unambiguously. Here, we used a hyperactivated RNA substrate bearing a 5'- phosphorothiolate to investigate the role of a critical cytosine residue in the hepatitis delta virus ribozyme. The hyperactivated substrate specifically suppressed the deleterious effects of cytosine mutations and pH changes, thereby linking the protonation of the nucleobase to leaving-group stabilization. We conclude that the active-site cytosine provides general acid catalysis, mediating proton transfer to the leaving group through a protonated N3-imino nitrogen. These results establish a specific role for a nucleobase in a ribozyme reaction and support the proposal that RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes.

For nearly two decades after the discovery of ribozymes, the view that divalent metal ions mediate RNA catalysis persisted, as all known ribozymes depended on metal ions for their function. The RNA nucleobases, in contrast, were considered poorly suited as catalytic agents. In recent years, structural and functional analyses of small ribozymes have challenged this view: not only do these ribozymes remain active in the absence of divalent metal ions, but their active sites contain nucleobases apparently poised to facilitate reaction chemistry1, 2, 3, 4, 5, 6, 7, 8. Even the ribosome may rely on direct nucleobase participation to mediate protein synthesis9, 10. These findings raised questions about the molecular strategies by which nucleobases mediate catalysis. For the hepatitis delta virus (HDV) ribozyme in particular, it has been proposed that a nucleobase may stabilize charge buildup in the transition state through proton transfer3, 4, 5.

The HDV ribozyme is a self-cleaving RNA motif that occurs in closely related genomic and antigenomic forms during the replication cycle of human HDV11. The self-cleavage reaction produces RNA containing 2',3'-cyclic phosphate and 5'-hydroxyl termini. Crystallographic analysis of the self-cleaved motif (product) has shown a critical cytosine (C76 and C75 as numbered in the antigenomic and genomic forms, respectively) at the active site, leading to the proposal that the nucleobase participates directly in catalysis1. Functional analyses have implicated C76 in proton transfer3, 4, 5, 11, 12, 13 fulfilling one of two kinetically equivalent roles—acting either as a general base3, 4 to deprotonate the 2'- OH (Fig. 1a) or as a general acid4, 5 to protonate the 5'-oxygen leaving group (Fig. 1b). Structural data support both possibilities: in the product, the cytosine N3 resides within hydrogen-bonding distance (2.7 Å) of the 5'-oxygen atom1; in the inactive C76U mutant 'precursor,' equal distances separate N3 (4.6−5.5 Å) from the 5'- and 2'-oxygen atoms14. However, global and local conformational changes accompany the cleavage reaction15, 16, 17, rendering uncertain the relationship between the active-site architectures shown in these structures and the transition-state architecture. Thus neither the structural nor the biochemical data have converged on a coherent mechanistic model for the role of the putative 'catalytic' cytosine nucleobase1, 3, 4, 5, 11, 14. We describe here an approach using a hyperactivated substrate that functionally links the ribozyme reaction center to the nucleobase and distinguishes between the kinetically equivalent18 roles proposed for the active-site nucleobase3, 4, 5, 11.

Figure 1: Kinetically equivalent roles proposed for the active-site cytosine in the HDV ribozyme reaction.

(a) Cytosine acts as a general base, mediating proton transfer from the 2'-hydroxyl3, 4, 14. (b) Alternatively, protonated cytosine acts as a general acid, mediating proton transfer to the 5'-oxygen leaving group4, 5. Both models include a hydrated magnesium ion implicated in the reaction5, 11, 37. The proton transfers associated with the 2'-OH and the 5'- oxygen may not be concerted. Further proposed interactions with the cytosine N4-amino group are not shown but have included the magnesium ion14, the nonbridging oxygen of the scissile phosphate13, 37 and other residues on the ribozyme1, 11.

Full figure and legend (23K) Figures, schemes & tables index

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Results

Hyperactivated RNA substrate S5'-S

Chemical perturbation of the leaving group in an enzyme-catalyzed reaction, in combination with enzyme active-site mutations, provides a means of identifying features that contribute to leaving-group stabilization, such as general acid catalysis or hydrogen- bond donation19, 20, 21, 22, 23, 24. An RNA oligonucleotide containing a 5'-bridging phosphorothiolate (5'-PS) linkage, in which a sulfur atom replaces the 5'-oxygen atom, offers such a biochemical probe for RNA and protein endonucleases. Under alkaline conditions, a 5'-PS linkage in RNA reacts nearly 105-fold faster than the native phosphodiester linkages, reflecting the greater acidity of the sulfur leaving group compared with the oxygen leaving group. This hyperactivated 5'-PS linkage shows susceptibility to base catalysis25, 26, reacting with a log-linear dependence on pH, but lacks susceptibility to general acid catalysis as a consequence of the weak hydrogen bond−accepting ability27 of the sulfur leaving group. Enzyme mutations or pH changes that disable donation of a hydrogen bond or proton to the leaving group during catalysis (such as those from a general acid) would be expected to affect the cleavage of the 5'-PS less adversely than cleavage of the native phosphodiester linkage. In contrast, mutations or pH changes that disable other catalytic features, including the action of a general base, would be expected to affect the 5'-PS and native phosphodiester cleavage reactions similarly.

To obtain an HDV substrate oligonucleotide containing a 5'-PS, we synthesized two nucleoside phosphoramidites for coupling by means of solid-phase chemistry—one containing the 5'-sulfur atom and the other containing a photolabile o-nitrobenzyl (oNBn)-protected 2'-hydroxyl group (Fig. 2a). To simplify the synthetic access, we chose a 2'-deoxynucleoside as the residue that bears the 5'-sulfur. After solid-phase oligonucleotide synthesis and deprotection, the vicinal 2'-hydroxyl remains protected as the oNBn ether, allowing experimental manipulation and radiolabeling of the oligonucleotide without degradation at the 5'-PS linkage (S.R.D. and J.A.P., unpublished data). We used the same 2'-O-oNBn phosphoramidite to construct the corresponding control oligonucleotide RNA containing the native phosphodiester linkage and 2'- deoxyguanosine. Benchtop irradiation (4 min with a 100-W UV (365-nm) lamp) furnished the fully deprotected RNA for immediate biochemical use28 (Fig. 2a). The two oligonucleotides, S5'-S and S5'-O, differing by the single 5'-bridging atom at the scissile phosphate, were tested as substrates for the HDV ribozyme.

Figure 2: The wild-type HDV ribozyme catalyzes cleavage of the hyperactivated substrate S5'-S.

(a) Synthetic nucleoside phosphoramidites for constructing the oligonucleotides containing a photolabile 2'-O-oNBn ether and a 2'-deoxyguanosine residue bearing either a 5'-oxygen or a 5'-sulfur. UV irradiation of the 5'-32P-labeled oligonucleotides produced the substrates S5'-O and S5'-S, respectively. (b) Sequence and secondary structure of the trans-HDV ribozyme (black/red) substrate (blue) complex used. The complex is derived from the natural self-cleaving antigenomic HDV motif11. Red letters indicate residues targeted for mutation (see Table 1), arrowheads in a and b mark the cleavage site, and g is the 5'-terminal guanosine included for efficient transcription. (c) The pH dependence of the cleavage rate constant kcleavage (see Methods). Circles indicate the rate constant for reactions of S5'-O (green) and S5'-S (blue) in the presence of saturating ribozyme, and light blue squares the rate constants for cleavage of S5'-S in the absence of ribozyme (10 mM MgCl2). See Methods for fit of these data. The data point at pH 9.9 for wild-type cleavage of S5'-S was not included in the fit. The dotted curve is the predicted profile for S5'-S cleavage, assuming the observed rate reflects the sum of the wild type ribozyme−catalyzed cleavage rate (with two-pKa pH dependence) and the 'background' (10 mM MgCl2) cleavage rate. (d) Cleavage response to increasing softness of the cation environment. Divalent ions can enhance the 'background' cleavage rate of S5'-S (light blue bars) but have no effect on the ribozyme-catalyzed cleavage of S5'-O (green bars) and S5'-S (blue bars) at pH 6.0. Individual rate constants were reproducible within 10%.

HDV ribozyme catalyzes cleavage of S5'-S

We used a kinetically well characterized trans-acting form of the HDV ribozyme11 based on the antigenomic sequence (Fig. 2b; referred to herein as 'wild-type'). In reactions with S5'-O, this ribozyme showed characteristics similar to those of other described antigenomic 15, 29 trans-HDV ribozymes : an apparent KM of 200 nM, a cleavage rate constant of 1 min-1 (under 'standard conditions': 10 mM Mg2+, pH 7.5) and a bell-shaped pH dependence (Fig. 2c; green circles). At pH 7.5 in the presence of the wild-type HDV 2+ ribozyme and 10 mM Mg , S5'-S reacted at nearly the same rate as S5'-O. Both reactions required Mg2+ and were affected by cobalt hexammine5 with nearly the same concentration dependence (data not shown). Moreover, reactions with S5'-S and S5'-O showed the same pH dependence in the pH range from 4.5 to 6.3, increasing log-linearly with unit slope. Over this pH range, S5'-S reacted three orders of magnitude faster than in the absence of ribozyme (extrapolated 'background'; Fig. 2c, light blue squares). These observations strongly suggest that the reaction of S5'-S reflects ribozyme-mediated catalysis rather than simply fortuitous enhancement of the background reaction upon hybridization to the ribozyme.

2+ Further observations distinguished the reaction of the catalytic complex E S5'-S nMg from the background reaction without ribozyme. (i) The cleavage of S5'-S in the presence of wild-type ribozyme showed no dependence on pH above 6.3, but the background reaction increased log-linearly over that pH range. (ii) In buffer containing 5 mM Mg2+, addition of softer divalent metal ions enhanced the background S5'-S cleavage, as observed previously25 (Fig. 2d; light blue bars), but provided no stimulation in the presence of ribozyme (Fig. 2d; blue bars). As softer divalent metal ions coordinate sulfur more 2+ effectively than does Mg , the absence of stimulation of the S5'-S reaction relative to S5'-O gives no indication that the leaving group interacts directly (through inner-sphere coordination) with a metal ion during the catalytic process. (iii) Cobalt hexammine had no effect on the background reaction (data not shown) but inhibited ribozyme-catalyzed cleavage of S5'-S (and S5'-O (ref. 5), as noted above).

The log-linear unit-slope stimulation shown by reactions of S5'-O and S5'-S catalyzed by the wild-type ribozyme when the pH is raised from 4.5 to 6.3 suggested that the catalytic E S nMg2+ complexes for both substrates benefit from a net proton loss before the cleavage event. The source of the proton and the mechanism by which it affects the reaction remain undefined, although several models have been proposed3, 4, 5 (Fig. 1a,b). Above pH 8, the pH-rate profiles diverge. The reaction rate of S5'-O decreased log-linearly with unit 2+ slope, suggesting that the net loss of a proton from the E S5'-O nMg complex deleteriously affects the reaction. In marked contrast, the reaction of S5'-S showed no 2+ dependence on pH in this region, suggesting either that E S5'-S nMg undergoes no net proton loss over this pH range or that proton loss occurs but has no effect on S5'-S cleavage. Thus the pH-induced inhibition observed for S5'-O cleavage was suppressed upon hyperactivation of the leaving group in S5'-S. Together these results imply that a proton, directly or indirectly, plays a role in leaving-group stabilization. The log-linear decline in S5'-O reactivity with increasing pH therefore may reflect depletion of a general acid in the ribozyme through deprotonation.

Hyperactivated substrate S5'-S rescues C76U defect

To probe the role of the active-site cytosine in catalysis, we constructed a mutant ribozyme containing uracil at residue 76 (C76U). In the presence of the C76U ribozyme 2+ 11, 30 and 10 mM Mg , S5'-O reacted at a speed below the limit of detection , whereas S5'-S reacted more than 46,000 times faster (Fig. 3a; Table 1) at pH 7.5, which is within a log- linear portion of the pH-rate profile (Fig. 3b). Moreover, the C76U ribozyme catalyzed 2+ S5'-S cleavage with the same Mg dependence (Fig. 3c) and showed the same cobalt hexammine inhibition (Fig. 3d) as the wild-type ribozyme. In contrast to the C76 mutation, mutations of other residues in the ribozyme core affected the cleavage rates of S5'-S and S5'-O similarly (Table 1). The observation that hyperactivation of the leaving group specifically suppressed the deleterious effect of the C76 mutation provides functional evidence that C76 is involved, directly or indirectly, in leaving-group activation.

Figure 3: S5'-S suppresses the C76U mutant defect.

(a,b) S5'-O undergoes no detectable cleavage with the C76U mutant ribozyme and 10 mM 2+ Mg (pH 7.5), whereas S5'-S reacts readily (a) and in a pH-dependent manner (b). Data were fit by linear regression and gave a slope of 0.96 0.05. (c) Magnesium dependence of cleavage of S5'-S by wild type (circles) and C76U (triangles) ribozymes. Rate constants are normalized to maximal cleavage activity. Fits of the data to the Hill equation knorm = 2+ n 2+ n n kmax [Mg ] /([Mg ] + K D,app) with unit Hill coefficient (n) yield apparent dissociation constant KD,app values of 8.5 1.3 mM (wild type; solid curve) and 8.4 1.3 mM (C76U; 3+ dotted curve). (d) Cobalt hexammine ([Co(NH3)6] ) inhibition of cleavage by wild type 2+ (circles) and C76U (triangles) of S5'-S (pH 7.5; 10 mM Mg ; k0 is the rate constant for 3+ cleavage in the absence of [Co(NH3)6] ). Fits to the data using a binding equation (as in c) gave apparent inhibition constant Ki,app values of 27.2 5.0 M for wild type (solid curve) and 19.5 1.5 M for C76U (dashed curve). (e) Imidazole buffer concentration dependence of C76U cleavage activity for S5'-S (blue) and S5'-O (green). Reactions were at pH 7.5 and contained 10 mM Mg2+. Data were fit by linear regression.

Table 1: Rate constants for the cleavage of S5'-O and S5'-S catalyzed by HDV ribozymes

Full table Figures, schemes & tables index

At pH 7.5, S5'-S seemed to suppress the C76U defect completely. However, at pH 6.0 and below, S5'-S suppression of the C76U defect was incomplete: reaction from the E S5'-S nMg2+ complex was two orders of magnitude slower than that from the corresponding wild-type complex (Fig. 3b, Table 1). We attempted to rescue the C76U defect with imidazole. Imidazole and its analogs have been shown to rescue the cleavage activity of the C76U ribozyme in a concentration-dependent manner3, 4. The rescue efficiency depends log-linearly on the pKa of the imidazole analog, which is consistent with the possibility that imidazole, and by implication C76, mediates proton transfer. We monitored the C76U-catalyzed reactions of S5'-O and S5'-S over a range of imidazole buffer concentrations at pH 7.5, which lies in the log-linear portion of the pH-rate profile of S5'-S cleavage (Fig. 3e). Increasing imidazole concentration progressively stimulated the 3, 4 C76U-catalyzed cleavage of S5'-O, as observed previously , but not the corresponding S5'- S reaction. Imidazole rescue of the S5'-O reaction but not the S5'-S reaction supports the possibility that S5'-S and imidazole use redundant mechanisms (activation of the leaving group) to suppress the C76U defect. This result and the previous inference that the rescuing imidazole mediates proton transfer3, 4 together support a model in which imidazole (and by implication C76) alleviates the C76U defect by providing general acid catalysis.

Crystallographic analysis of the genomic precursor bearing the C76U mutation showed electron density for a hydrated cation at the active site, located within outer-sphere coordination distance of the 5'-oxygen leaving group. This raised the possibility that a metal ion−bound water molecule might donate a proton to the leaving group during 14 catalysis . We used reactions with S5'-S in the presence of cobalt hexammine to probe this apparent link between the leaving group and the metal ion. Cobalt hexammine, an exchange-inert metal complex that binds to outer-sphere coordination sites in RNA, inhibits the HDV ribozyme reaction in a concentration-dependent manner, putatively by competing with a hydrated magnesium ion for binding at the active site5. Consistent with this hypothesis, cobalt hexammine occupies the divalent cation binding site in the C76U mutant structure14. In contrast to magnesium hexahydrate, the cobalt hexammine complex presents ligands that bear neither available lone-pair electrons nor exchangeable protons, rendering cobalt hexammine−mediated proton transfer highly unlikely. If cobalt hexammine inhibits the reaction solely by blocking magnesium hexahydrate mediated proton transfer to the leaving group, then hyperactivation of the leaving group would be expected to suppress cobalt hexammine inhibition. As noted above, however, cobalt hexammine inhibited cleavage reactions of S5'-S mediated by both wild-type and C76U ribozymes (Fig. 3d) and did so with the same concentration dependence as was observed for S5'-O cleavage. These observations provided no evidence to link the leaving group to the metal ion (see also Fig. 2d). However, we cannot exclude the possibility that the inhibition of the S5'-S reaction results either from a shift in the active-site architecture upon cobalt hexammine binding31 or from the inability of cobalt hexammine to mimic further roles of Mg2+ in catalysis.

Functional linkage of leaving group and C76 ring nitrogen

The crystal structure of the HDV ribozyme self-cleavage product suggests that the 'catalytic' cytosine N4-amino group engages in a network of hydrogen bonds1. The absence of this amino group in the C76U mutant may impair catalytic interactions other than those associated with leaving-group activation, possibly accounting for the mutant's defective cleavage of S5'-S. Consequently, we further explored the apparent link between C76 and the leaving group by constructing a mutant ribozyme containing 3-deazacytosine (c3C) in lieu of C76. This mutation, which replaces the N3-imino nitrogen of cytosine with a methine group, retains the N4-amino group but eliminates the possibility of proton transfer through N3. We synthesized the heterocycle c3C, from which we generated the riboside and then transformed it to the corresponding phosphoramidite derivative for incorporation into an oligonucleotide (Fig. 4a)32, 33, 34. Ligation of the synthetic oligonucleotide to a transcribed RNA strand35, 36 furnished the full-length mutant ribozyme C76c3C (Fig. 4a).

Figure 4: The single-atom-mutant ribozyme C76c3C shows nearly wild-type activity against S5'-S. (a) Phosphoramidite synthesis and splint ligation strategy for construction of the C76c3C ribozyme. Asterisks mark the ligation site and the circle within the secondary structure 2+ highlights the position of c3C. (b) Mg dependence of C76c3C-catalyzed cleavage of S5'- S (pH 7.5). The data are fit by the Hill equation (see Fig. 3c) with unit Hill coefficient giving an apparent dissociation constant KD,app value of 44.0 11.2 mM (pH 7.5). (c) Catalytic deficiency induced by the C76c3C mutation relative to the wild-type ribozyme. The relative reactivity of the C76U mutant is shown for comparison. Boxes on the nucleobase structure highlight the specific perturbation(s) in the mutants. Reactions were 2+ at pH 5.5, which is in the log-linear region for reactions of S5'-S up to 110 mM Mg , and rate constants were reproducible within 10%. We could not obtain reliable rate constants for reactions that included more than 110 mM Mg2+ at pH 5.5.

At pH 5.5 (in the log-linear pH regime) and 10 mM Mg2+, C76c3C catalyzed the cleavage of S5'-S about an order of magnitude faster than did C76U, achieving an efficiency within 17-fold of that of the wild-type ribozyme. These results indicate that the N4-amino group of C76 contributes to HDV catalysis, consistent with implications from previous studies10, 12, 37. To enable comparison of C76c3C, C76U and the wild-type ribozymes under conditions of saturating Mg2+, we examined the Mg2+ dependence of the C76c3C- 2+ mediated S5'-S reaction. Mg stimulated the C76c3C-mediated reaction with unit Hill coefficient but with a higher apparent dissociation constant (Fig. 4b) than was observed for the wild-type- and C76U-ribozyme reactions. In the presence of 110 mM Mg2+, the C76c3C mutant catalyzed cleavage of S5'-S only fourfold more slowly than did the wild- type ribozyme (Fig. 4c). In marked contrast to the reaction with S5'-S, C76c3C did not -6 -1 show catalytic activity against S5'-O (that is, <6.9 10 min detection limit) under any of the conditions tested. Thus, a single-atom mutation in the ribozyme abolished detectable activity when the substrate bore an oxygen leaving group but retained almost full activity when the substrate bore the hyperactivated sulfur leaving group. This nearly quantitative suppression of the C76c3C defect by S5'-S strengthens the link between the leaving group and C76 and directly implicates the C76 N3 ring nitrogen.

6-Azacytosine and S5'-S implicate C76 in general acid catalysis

Having established a link between the leaving group and the N3 nitrogen atom of C76, we explored the relationship between this active-site residue and the pH-rate profile. The 'bell-shaped' pH-rate profile for S5'-O cleavage by the wild-type ribozyme indicates that at least two distinct titrations govern the reaction rate—one that stimulates it (Fig. 2c; acidic limb of green curve) and one that inhibits it (Fig. 2c; basic limb of green curve). Two kinetically equivalent models can account for this pH dependence (Fig. 5)18. In one model, the pKa of the rate-stimulating titration (Fig. 5a; solid blue curve) lies below the pKa of the rate-inhibiting titration (Fig. 5a; solid red curve) such that the two deprotonations occur in distinct regions of the pH profile (distinct titrations). In the second model, the pKa of the rate-stimulating titration (Fig. 5b; solid blue curve) lies above the pKa of the rate-inhibiting titration (Fig. 5b; solid red curve) such that both deprotonations occur partially in the same region of the pH profile (overlapping titrations). Both models would result in a bell-shaped profile (Fig. 5c,d; solid black curves)18. The locations of these deprotonations within the E S nMg2+ complex and the mode by which they stimulate or inhibit catalysis remain unclear. To test the possibility that C76 itself undergoes deprotonation over the pH range examined, we examined the effect of a pKa-perturbing mutation on the pH dependence. In practice, this required a cytosine mutation that retains some activity against S5'-O. We chose 6-azacytosine (z6C) for this purpose; the free nucleoside analog ionizes at N3 with a lower pKa of 2.6 (ref. 38), compared with 4.2 for cytidine, and interferes weakly with HDV activity when incorporated into C75 (the genomic HDV equivalent of C76) through transcription13. We synthesized 6-azacytidine and its protected phosphoramidite analog39 and incorporated it into the full-length ribozyme C76z6C by solid-phase synthesis and splint ligation (as described for C76c3C).

Figure 5: Kinetically equivalent models from two independent, offsetting titrations simulate the pH dependence of the HDV ribozyme.

(a−d) The titrations may occur (a) in distinct regions of pH or (b) in overlapping regions of pH, leading to the same bell-shaped pH dependence (solid black curves) in c and d, respectively. The analysis assumes that the ribozyme bears two titratable groups whose pKa values differ by two units ( pKa = |pKa1 - pKa2| = 2). Deprotonation of one group increases the fraction of fully functional ribozyme (blue curves = rate-stimulating titration), whereas deprotonation of the other group decreases the fraction (red curves = rate-inhibiting titration). The dashed blue and red curves simulate the effect of a mutation that decreases the lower pKa by two units (pK'a1 or pK'a2), resulting in broadening of the pH-independent region for the fraction of fully functional ribozyme (dashed black curves in c and d). See Methods for details of simulations. Further possible but unlikely modes by which a wider pH-independent region may result from decreasing the higher pKa are discussed in the Supplementary Note online.

We obtained the pH-rate profile for cleavage of S5'-O by the C76z6C mutant ribozyme in the presence of 10 mM Mg2+. The C76z6C ribozyme showed at pH 7.5 the same Mg2+ dependence as the wild-type and C76U ribozymes—an apparent dissociation constant of 10.3 1.1 mM with unit Hill coefficient (data not shown). Compared with the reaction catalyzed by wild type, the C76z6C-catalyzed reaction of S5'-O showed a wider pH- independent region (Fig. 6; green circles), reflecting a larger apparent pKa between the titrations. We analyzed this change in pH-rate profile in terms of the models described by distinct titrations or overlapping titrations. When distinct titrations (Fig. 5a,c) govern a reaction, a wider pH-independent region (Fig. 5c; dashed black line) could result from a mutation that lowers the pKa of the rate-stimulating titration (Fig. 5a; dashed blue line). Alternately, when overlapping titrations (Fig. 5b,d) govern a reaction, a wider pH- independent region (Fig. 5d; dashed black line) could result from a mutation that lowers the pKa of the rate-inhibiting titration (Fig. 5b; dashed red line). Thus the pKa-lowering z6C analog at residue 76 shifts the pH-rate profile in a manner expected from decreasing the pKa of a titration that governs the rate, supporting previous proposals that C76 itself bears one of the titratable groups3, 4, 5, 11. However, because both models account for the observed profile, we cannot determine whether the z6C mutation affects the rate- stimulating or the rate-inhibiting titration. This ambiguity underscores the limitations of kinetic analysis using the native RNA substrate S5'-O alone.

Figure 6: The pH dependence of the C76z6C mutant ribozyme implicates C76 in general acid catalysis.

The pH-rate profiles are shown for C76z6C-catalyzed cleavage of S5'-O (green circles) and S5'-S (blue circles). Reaction conditions and fits to the data are described in Methods. The structure of the nucleobase 6-azacytosine with pyrimidine numbering is shown. In the free nucleoside, the iminium ion bearing a proton at N3 ionizes with a lower pKa of 2.6 as compared with 4.2 for cytidine13, 38.

To resolve this ambiguity, we compared the pH dependence for the C76z6C-catalyzed reactions of S5'-O and S5'-S (Fig. 6; green and blue circles, respectively). The reaction rate of S5'-O increased with pH and became pH independent above pH 6. This transition in the pH dependence may reflect (i) a change in the rate-determining step from a pH- dependent step to a pH-independent conformational change, (ii) the pKa of the rate- stimulating titration (Fig. 5a) or (iii) the onset (pKa) of the rate-inhibiting titration that offsets the rate-stimulating titration (Fig. 5b). The possibility that a pH-independent conformational change limits the reaction above pH 6 seems small, as the reaction of the 2+ E S5'-S nMg complex continued to increase with pH, becoming several orders of 2+ magnitude faster than the corresponding reaction of the E S5'-O nMg complex. This divergence in pH dependence between S5'-S and S5'-O presumably occurred because the S5'-S reaction is sensitive only to the rate-stimulating titration and not to the rate-inhibiting titration, whereas the S5'-O reaction is sensitive to both. Thus the point of divergence in the S5'-O and S5'-S profiles signals the onset of the rate-inhibiting titration. If the profiles diverge at the higher pKa of the S5'-O profile, then distinct titrations must govern HDV catalysis (Fig. 5a). In contrast, if the profiles diverge at the lower pKa of the S5'-O profile, then overlapping titrations must govern HDV catalysis (Fig. 5b). The observed reactions of S5'-O and S5'-S with C76z6C ribozyme clearly diverged at the lower pKa, strongly suggesting that overlapping titrations govern catalysis by the C76z6C ribozyme. This result, together with the z6C-induced widening of the pH-independent region, supports a model in which the rate-inhibiting titration in the HDV ribozyme corresponds to deprotonation at C76. If deprotonation of z6C inhibits the reaction rate, it follows that protonation of z6C allows function. This finding, and the established link between the cytosine N3 ring nitrogen and the leaving group, together support a model in which the active-site cytosine provides general acid catalysis.

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Discussion

We have shown that hyperactivation of the leaving group in the HDV-ribozyme reaction resulted in a chemogenetic suppression of (i) the effect of the rate-inhibiting deprotonation on the wild-type and C76z6C-mutant ribozymes, (ii) the lethality of C76U and C76c3C mutations, but not the deleterious effect of other core mutations or cobalt hexammine inhibition, and (iii) the rescuing effect of imidazole-mediated proton transfer on the C76U mutant ribozyme. Furthermore, this hyperactivated substrate in combination with the pKa-lowering C76z6C mutation links the active-site cytosine to a titration that inhibits the reaction rate. These observations suggest that the active-site cytosine acts as a general acid in the HDV ribozyme reaction, donating a proton to the leaving group through N3. Although indirect effects could possibly give rise to the observed functional relationship and may account for individual observations, the general acid model for cytosine-mediated catalysis (Fig. 1b)5 accounts for all the data. These findings bolster the proposal that RNA nucleobases may function in a manner analogous to catalytic histidine residues in protein enzymes1, 3, 5.

Nucleobase catalysis has been proposed in other natural nucleolytic ribozymes2, 6, 7, 8, 40, 41. It may be operative in the recently discovered regulatory ribozyme42 and more generally in nucleic acid enzymes that emerge from in vitro selection. However, there are no functional data that link nucleobases to the reaction center in these enzymes. As described here, coupling substrate hyperactivation with sequence and atomic mutations provides a strategy both to establish such a linkage and to distinguish between kinetically equivalent mechanisms that commonly confound mechanistic analyses of acid-base catalysis.

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Chemical synthesis.

The synthesis of the two phosphoramidites, and the subsequent solid-phase synthesis and deprotection to furnish the oligonucleotides S5'-O and S5'-S in 'caged' (2'-O-oNBn) form, will be reported elsewhere. After the synthesis and deprotection protocols, the unpurified 'caged' S5'-S was identified by MALDI mass spectrometry (PE Biosystems). The observed mass (3,637.58) was consistent with the expected increase in mass from the unmodified undecamer substrate RNA (3,503.92) prepared and deprotected under similar conditions. After denaturing polyacrylamide gel purification, the S5'-S oligomer was quantitatively cleaved by AgNO3 at the expected PS linkage, indicating the homogeneity of the S5'-S substrate. After UV deprotection of the caging group, the kinetics of the cleavage of the 5'-PS−containing RNA by divalent metal ions were similar to those reported previously for the 5'-PS linkage25.

The 3-deazacytidine phosphoramidite derivative was synthesized beginning with the transformation of 4-amino-2-chloropyrimidine to the corresponding pyridone32. Silylation and Vorbrüggen coupling to 1-O-acetyl-2,3,5-tri-O-benzoyl- -D-ribofuranose followed by deprotection yielded 3-deazacytidine33. The N4-amino group was protected as the phenoxyacetamide (for which quantitative deprotection by NH4OH at 55 °C was complete within 2 h), the 5'-hydroxyl as the dimethoxytrityl ether and the 2'-hydroxyl as the t-butyldimethylsilyl ether in successive steps by standard procedures (see Supplementary Fig. 1 online for 1H-NMR). Phosphitylation with 2-cyanoethyl diisopropyl chlorophosphoramidite provided the target synthon for solid-phase oligoribonucleotide synthesis (see also ref. 34).

The 6-azacytidine derivative was obtained from 6-azauridine (Sigma); appropriate protecting and functional groups for solid-phase synthesis were incorporated in subsequent steps39 (see Supplementary Fig. 2 online for 1H-NMR).

Ribozyme preparation.

The HDV wild-type and site-specific mutant ribozymes (sequence in Fig. 2b) were prepared by T7 RNA polymerase transcription from the single-stranded DNA template (IDT) with the requisite promoter sequence using standard procedures43. The ribozyme is derived from the natural self-cleaving antigenomic HDV motif and contains a shortened P4 stem and a discontinuous J1/2 region. The numbering (see Fig. 2b), with discontinuity in the P4 stem, follows that established for the antigenomic ribozyme11. For efficient transcription, the ribozyme contains a 5'-terminal guanosine (Fig. 2b) rather than the natural AU sequence. For the single-atom mutant ribozymes C76c3C and C76z6C, we synthesized the oligoribonucleotides 5'-UGGCUAAGGGAGAGCCA-3' (where C represents the modified nucleotide) on a Millipore solid-phase DNA/RNA synthesizer, then deprotected and purified the oligonucleotide using standard procedures. This synthesized RNA corresponds to the last 17 nucleotides at the 3' end of the ribozyme and was ligated35, 36 using a DNA splint (5'- GTGGCTCTCCCTTAGCCATCCGAGTGCTCGGATGC-3') to the 50-nucleotide 5' sequence (see Fig. 4) obtained by transcription. The transcription of the 5' sequence was carried out with a DNA template with the last two nucleotides 2'-methoxylated to ensure 3'-end homogeneity44. A control RNA corresponding to the wild-type sequence was also obtained by similar ligation. Over the pH range 5.5−9.6, the catalytic activity of this 'ligated wild-type' ribozyme varied from that of the transcribed wild-type ribozyme by less than 5%, suggesting that the extra experimental procedures used to obtain the ligated ribozyme have negligible effects.

Ribozyme reactions and analyses.

Ribozymes were preincubated with 5−10 mM MgCl2 for 2 min at 70 °C followed by 14 min at 25 °C; then appropriate buffer was added and they were incubated a further 60 s. Irradiation by a 100-W UV lamp (365 nM) for 4 min (corresponding to 10 half-lives) removed the 2'-O-oNBn group on the 5'-radiolabeled substrate (2−5 l). Reaction was initiated by addition of the substrate to the ribozyme and metal solution and incubation at 25 °C for the duration of the experiment. All reactions described contain saturating ribozyme concentrations, such that the observed rate constants reflect the cleavage rates of a single turnover from the enzyme-substrate complex (E S nMg2+). Reactions typically consisted of 1 M ribozyme (control reactions with two- to fivefold higher ribozyme concentrations under various pH and divalent-ion concentration regimes show a 1 M ribozyme concentration to be saturating), trace (<5 nM) substrate RNA, appropriate divalent metal, 50 mM sodium MOPS (NaMOPS) buffer (pH 6.5, 7.5), or, for pH dependence, buffer containing either 25 mM MES, 25 mM acetic acid and 50 mM Tris (pH 4.0−8.0), or 50 mM MES, 25 mM Tris and 25 mM 2-amino-2-methyl-1- propanol (pH 8.0−9.5)3. Aliquots of 1.5−2 l were removed at appropriate intervals, quenched in 6−8 l of 90% formamide and 20 mM EDTA, placed immediately on dry ice and, when necessary, stored overnight at -80 °C. The 5'-labeled cleavage product was separated from the uncleaved substrate by denaturing 20% polyacrylamide, 8 M urea gel electrophoresis. The cleavage products were quantified and normalized to the sum of the substrate and product bands with a PhosphorImager with ImageQuant software (Molecular Dynamics). The data for cleavage reactions were fit by the equation y = y0 + -k t ae cleavage with SigmaPlot software (Systat Software), where kcleavage is the first-order rate constant for cleavage. pH-reactivity curve fitting and simulation.

The rate constants for wild-type- and C76z6C-catalyzed cleavage of S5'-O (Figs. 2c and 6; pH-pKa1 pKa2-pH pKa2-pKa1 green circles) were fit by the equation kcleavage = kmax/(1 + 10 + 10 + 10 ), which yielded apparent pKas of 6.3 0.1 and 8.6 0.1 (wild type; green curve in Fig. 2c) and 6.1 0.2 and 9.4 0.1 (C76z6C; green curve in Fig. 6). The rate constants for cleavage of S5'-S by wild-type and C76z6C ribozymes (Figs. 2c and 6; blue circles) were pH-pKa1 fit by the equation kcleavage = kmax/(1 + 10 ), which yielded the apparent pKas 6.4 0.1 and 8.4 0.1, respectively (Figs. 2d and 6; blue curves). For cleavage of S5'-O and S5'-S by the wild-type ribozyme, the pKas are kinetic pKas. Thus the profiles of S5'-S and S5'-O reactions with the wild-type ribozyme diverge at the higher pKa because a conformational change limits the reaction rate in the pH-independent region (S.R.D. and J.A.P., unpublished data). The pKa for the C76n6C reaction against S5'-S is also a kinetic pKa. For C76z6C cleavage reactions of S5'-O, the pKas are likely to reflect those of the titrating groups; however, the higher pKa may be ill defined, as it lies close to the pH at which RNA denaturation might become a competing process.

The pH dependence of the rate-stimulating titration (blue curves) and the rate-inhibiting titration (red curves) in Figures 5a,b are simulated as f = 1/(1 + 10pKa1-pH) and f = 1/(1 + 10pH-pKa2), respectively. The fraction of fully functional enzyme affected by both independent titrations is simulated as f = 1/(1 + 10pH-pKa1 + 10pKa2-pH + 10pKa2-pKa1) in Figure 5c,d. The simulations do not take into account effects arising from possible cooperativity between the two titrations (see ref. 5). Such effects may influence the pKas, but not the trends.

Accession codes.

BIND identifiers (http://bind.ca/): 261927, 261928.

Note: Supplementary information is available on the Nature Chemical Biology website.

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Acknowledgments

We thank A. Ke and J.A. Doudna for helpful discussions and J. Staley, M.D. Been, P.C. Bevilacqua, D. Herschlag and members of the Piccirilli laboratory for critical comments and suggestions on the manuscript. We also thank J. Olvera for T4 DNA ligase and E. Duguid for assistance with MALDI mass spectroscopy. This work was supported by the Howard Hughes Medical Institute.

Competing interests

The authors declared no competing interests.

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References

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Top of page 1. Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology and Chemistry, University of Chicago, 5841 S. Maryland Avenue, MC1028, Chicago, Illinois 60637, USA. 2. Email: [email protected]

Correspondence to: Joseph A Piccirilli1 Email: [email protected]

(a) Cytosine acts as a general base, mediating proton transfer from the 2'-hydroxyl3, 4, 14. (b) Alternatively, protonated cytosine acts as a general acid, mediating proton transfer to the 5'-oxygen leaving group4, 5. Both models include a hydrated magnesium ion implicated in the reaction5, 11, 37. The proton transfers associated with the 2'-OH and the 5'- oxygen may not be concerted. Further proposed interactions with the cytosine N4-amino group are not shown but have included the magnesium ion14, the nonbridging oxygen of the scissile phosphate13, 37 and other residues on the ribozyme1, 11. (a) Synthetic nucleoside phosphoramidites for constructing the oligonucleotides containing a photolabile 2'-O-oNBn ether and a 2'-deoxyguanosine residue bearing either a 5'-oxygen or a 5'-sulfur. UV irradiation of the 5'-32P-labeled oligonucleotides produced the substrates S5'-O and S5'-S, respectively. (b) Sequence and secondary structure of the trans-HDV ribozyme (black/red) substrate (blue) complex used. The complex is derived from the natural self-cleaving antigenomic HDV motif11. Red letters indicate residues targeted for mutation (see Table 1), arrowheads in a and b mark the cleavage site, and g is the 5'-terminal guanosine included for efficient transcription. (c) The pH dependence of the cleavage rate constant kcleavage (see Methods). Circles indicate the rate constant for reactions of S5'-O (green) and S5'-S (blue) in the presence of saturating ribozyme, and light blue squares the rate constants for cleavage of S5'-S in the absence of ribozyme (10 mM MgCl2). See Methods for fit of these data. The data point at pH 9.9 for wild-type cleavage of S5'-S was not included in the fit. The dotted curve is the predicted profile for S5'-S cleavage, assuming the observed rate reflects the sum of the wild type ribozyme−catalyzed cleavage rate (with two-pKa pH dependence) and the 'background' (10 mM MgCl2) cleavage rate. (d) Cleavage response to increasing softness of the cation environment. Divalent ions can enhance the 'background' cleavage rate of S5'-S (light blue bars) but have no effect on the ribozyme-catalyzed cleavage of S5'-O (green bars) and S5'-S (blue bars) at pH 6.0. Individual rate constants were reproducible within 10%. (a,b) S5'-O undergoes no detectable cleavage with the C76U mutant ribozyme and 10 mM 2+ Mg (pH 7.5), whereas S5'-S reacts readily (a) and in a pH-dependent manner (b). Data were fit by linear regression and gave a slope of 0.96 0.05. (c) Magnesium dependence of cleavage of S5'-S by wild type (circles) and C76U (triangles) ribozymes. Rate constants are normalized to maximal cleavage activity. Fits of the data to the Hill equation knorm = 2+ n 2+ n n kmax [Mg ] /([Mg ] + K D,app) with unit Hill coefficient (n) yield apparent dissociation constant KD,app values of 8.5 1.3 mM (wild type; solid curve) and 8.4 1.3 mM (C76U; 3+ dotted curve). (d) Cobalt hexammine ([Co(NH3)6] ) inhibition of cleavage by wild type 2+ (circles) and C76U (triangles) of S5'-S (pH 7.5; 10 mM Mg ; k0 is the rate constant for 3+ cleavage in the absence of [Co(NH3)6] ). Fits to the data using a binding equation (as in c) gave apparent inhibition constant Ki,app values of 27.2 5.0 M for wild type (solid curve) and 19.5 1.5 M for C76U (dashed curve). (e) Imidazole buffer concentration dependence of C76U cleavage activity for S5'-S (blue) and S5'-O (green). Reactions were at pH 7.5 and contained 10 mM Mg2+. Data were fit by linear regression. (a) Phosphoramidite synthesis and splint ligation strategy for construction of the C76c3C ribozyme. Asterisks mark the ligation site and the circle within the secondary structure 2+ highlights the position of c3C. (b) Mg dependence of C76c3C-catalyzed cleavage of S5'- S (pH 7.5). The data are fit by the Hill equation (see Fig. 3c) with unit Hill coefficient giving an apparent dissociation constant KD,app value of 44.0 11.2 mM (pH 7.5). (c) Catalytic deficiency induced by the C76c3C mutation relative to the wild-type ribozyme. The relative reactivity of the C76U mutant is shown for comparison. Boxes on the nucleobase structure highlight the specific perturbation(s) in the mutants. Reactions were 2+ at pH 5.5, which is in the log-linear region for reactions of S5'-S up to 110 mM Mg , and rate constants were reproducible within 10%. We could not obtain reliable rate constants for reactions that included more than 110 mM Mg2+ at pH 5.5.

(a−d) The titrations may occur (a) in distinct regions of pH or (b) in overlapping regions of pH, leading to the same bell-shaped pH dependence (solid black curves) in c and d, respectively. The analysis assumes that the ribozyme bears two titratable groups whose pKa values differ by two units ( pKa = |pKa1 - pKa2| = 2). Deprotonation of one group increases the fraction of fully functional ribozyme (blue curves = rate-stimulating titration), whereas deprotonation of the other group decreases the fraction (red curves = rate-inhibiting titration). The dashed blue and red curves simulate the effect of a mutation that decreases the lower pKa by two units (pK'a1 or pK'a2), resulting in broadening of the pH-independent region for the fraction of fully functional ribozyme (dashed black curves in c and d). See Methods for details of simulations. Further possible but unlikely modes by which a wider pH-independent region may result from decreasing the higher pKa are discussed in the Supplementary Note online. The pH-rate profiles are shown for C76z6C-catalyzed cleavage of S5'-O (green circles) and S5'-S (blue circles). Reaction conditions and fits to the data are described in Methods. The structure of the nucleobase 6-azacytosine with pyrimidine numbering is shown. In the free nucleoside, the iminium ion bearing a proton at N3 ionizes with a lower pKa of 2.6 as compared with 4.2 for cytidine13, 38.