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 N4amino 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 hydrogenbond 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 S 5'-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%.
Full figure and legend (61K) Figures, schemes & tables index
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 trans-HDV ribozymes15, 29: 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
ribozyme and 10 mM Mg2+, 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.
Further observations distinguished the reaction of the catalytic complex E S5'-S nMg2+
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 effectively than does Mg2+, 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 slope, suggesting that the net loss of a proton from the E S5'-O nMg2+ complex
deleteriously affects the reaction. In marked contrast, the reaction of S5'-S showed no
dependence on pH in this region, suggesting either that E S5'-S nMg2+ 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
and 10 mM Mg2+, S5'-O reacted at a speed below the limit of detection11, 30, whereas S5'-S
reacted more than 46,000 times faster (Fig. 3a; Table 1) at pH 7.5, which is within a loglinear portion of the pH-rate profile (Fig. 3b). Moreover, the C76U ribozyme catalyzed
S5'-S cleavage with the same Mg2+ 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
Mg2+ (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 =
kmax [Mg2+]n/([Mg2+]n + KnD,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;
dotted curve). (d) Cobalt hexammine ([Co(NH3)6]3+) inhibition of cleavage by wild type
(circles) and C76U (triangles) of S5'-S (pH 7.5; 10 mM Mg2+; k0 is the rate constant for
cleavage in the absence of [Co(NH3)6]3+). 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.
Full figure and legend (57K) Figures, schemes & tables index
Table 1: Rate constants for the cleavage of S5'-O and S5'-S catalyzed by HDV ribozymes
Full tableFigures, 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
C76U-catalyzed cleavage of S5'-O, as observed previously3, 4, 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
catalysis14. 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
highlights the position of c3C. (b) Mg2+ 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
at pH 5.5, which is in the log-linear region for reactions of S5'-S up to 110 mM Mg2+, 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.
Full figure and legend (64K) Figures, schemes & tables index
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-mediated S5'-S reaction. Mg2+ 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 show catalytic activity against S5'-O (that is, <6.9 10-6 min-1 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.
Full figure and legend (13K) Figures, schemes & tables index
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 pHindependent 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 pHindependent 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 ratestimulating 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.
Full figure and legend (13K) Figures, schemes & tables index
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 pHdependent step to a pH-independent conformational change, (ii) the pKa of the ratestimulating 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
E S5'-S nMg2+ complex continued to increase with pH, becoming several orders of
magnitude faster than the corresponding reaction of the E S5'-O nMg2+ 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.
Top of page
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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Methods
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-1propanol (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 +
ae-kcleavaget 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;
green circles) were fit by the equation kcleavage = kmax/(1 + 10pH-pKa1 + 10pKa2-pH + 10pKa2-
pKa1
), 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 fit by the equation kcleavage = kmax/(1 + 10pH-pKa1), 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.
Top of page
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.
Top of page
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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: jpicciri@uchicago.edu
Correspondence to: Joseph A Piccirilli1 Email: jpicciri@uchicago.edu
(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 N4amino 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
Mg2+ (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 =
kmax [Mg2+]n/([Mg2+]n + KnD,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;
dotted curve). (d) Cobalt hexammine ([Co(NH3)6]3+) inhibition of cleavage by wild type
(circles) and C76U (triangles) of S5'-S (pH 7.5; 10 mM Mg2+; k0 is the rate constant for
cleavage in the absence of [Co(NH3)6]3+). 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
highlights the position of c3C. (b) Mg2+ 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
at pH 5.5, which is in the log-linear region for reactions of S5'-S up to 110 mM Mg2+, 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.