General acid-base catalysis mediated by nucleobases in the hairpin ribozyme

Article abstract of DOI:10.1021/ja3067429

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pK_a. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pK_a consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.

Full text of DOI:10.1021/ja3067429

Article
pubs.acs.org/JACS
General Acid−Base Catalysis Mediated by Nucleobases in the Hairpin
Ribozyme
Stephanie Kath-Schorr,‡ Timothy J. Wilson,‡ Nan-Sheng Li,§ Jun Lu,§ Joseph A. Piccirilli,§
and David M. J. Lilley*,‡
‡Cancer Research UK, Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee
DD1 5EH, U.K.
§Departments of Biochemistry and Molecular Biology, and Chemistry, Gordon Center for Integrative Science, 929 East 57th Street,
The University of Chicago, Chicago 60637, United States
S
* Supporting Information
ABSTRACT: The catalytic mechanism by which the hairpin ribozyme accelerates
cleavage or ligation of the phosphodiester backbone of RNA has been incompletely
understood. There is experimental evidence for an important role for an adenine
(A38) and a guanine (G8), and it has been proposed that these act in general acid−
base catalysis. In this work we show that a large reduction in cleavage rate on
substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-
oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better
leaving group. This is consistent with A38 acting as the general acid in the
unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P
ribozyme increases with pH log−linearly, indicative of a requirement for a
deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first
increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base
catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude
that general acid−base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the
experimental data.
that multiple processes contribute to the overall rate enhance-
ment.
INTRODUCTION
■
RNA-mediated catalysis is important in biologically significant
RNA processing events and in the condensation of amino acids
in the ribosome.1 Contemporary ribozymes also offer a glimpse
of how phosphoryl transfer reactions might have been catalyzed
in an early ‘RNA world’, postulated to have played a key role in
the early development of life on the planet.2 Yet the chemical
mechanisms of even the relatively simple ribozymes presently
known to exist are incompletely understood and are sometimes
the subject of considerable debate.
The nucleolytic ribozymes are a group of five species that
carry out a site-specific cleavage or ligation of the
phosphodiester backbone of RNA in order to process
replication intermediates or transcripts or to control gene
expression. Despite sharing little similarity in sequence or
structure, these ribozymes each bring about cleavage by
nucleophilic attack of an O2′ on the adjacent phosphorus
atom (Figure 1A).
In principle the reaction could be accelerated by transition
state stabilization (e.g., by hydrogen bonding, or by electrostatic
stabilization of the formally dianionic transition state), general
acid−base catalysis (enhancing hydroxyl nucleophilicity by
deprotonation, and stabilization of the oxyanion leaving group
by protonation), or by conformational facilitation of the
trajectory into an in-line transition state, and it is quite possible
In this contribution we address the catalytic mechanism of
the hairpin ribozyme, derived from the (-) strand of the tobacco
ringspot virus satellite RNA.5,6 The secondary structure of the
ribozyme−substrate complex7 is based upon a four-way helical
junction (Figure 1B).8−10 Adjacent arms (A and B) contain
internal loops that undergo intimate interaction to generate the
active conformation of the ribozyme.11−13 Within these loops,
two nucleotides have been identified as particularly critical to
catalytic activity, G8 in loop A on the opposite strand to the
scissile phosphate,11−13 and A38 in loop B.14 We have
previously noted15 that the arrangement of these components
is very similar to that in the Varkud satellite ribozyme, where
there is good evidence that the corresponding guanine (G638)
acts as general base and the adenine (A756) as general acid in
the cleavage reaction.16 Crystal structures of the hairpin
ribozyme both in the four-way junction form17 and the
minimal hinged version (lacking helices C and D)18,19 show
that G8 and A38 are close to the scissile phosphate, and in the
structure of a transition-state analogue20 A38 and G8 are
hydrogen bonded to O5′ and O2′, respectively, thus positioned
Received: July 11, 2012
Published: September 7, 2012
© 2012 American Chemical Society
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general base or by other mechanisms such as transition state
stabilization, the restorative effect of the 5′-PS substitution
should be much smaller. In addition, if the general acid no
longer carries out its catalytic function in the presence of the 5′-
PS group, the general base can then be studied in isolation. We
critically examined the role of A38 and G8 in the hairpin
ribozyme by this approach. The results are consistent with their
proposed function in general acid−base catalysis.
EXPERIMENTAL SECTION
■
The chemical synthesis of A[2′-O-(o-nitrobenzyl)]-psG is described in
the Supporting Information and Scheme S1. A substrate strand
containing this dinucleotide was prepared by ligation to the 5′- and 3′-
oligonucleotides. The hairpin ribozyme was constructed by hybrid-
ization of four oligonucleotides as shown in Figure S1 (SI). All
methods and sequences are fully described in the SI.
Ribozyme Kinetics. Cleavage of the radioactively 5′-32P-labeled
substrate strand was studied under single-turnover conditions as
described in Wilson et al.16 The 2-hydroxyl of the adenosine was
deprotected using UV irradiation for 15 min under neutral conditions.
Following equilibration to 25 °C, reactions were started by adding
MgCl2. Standard reaction conditions for the hairpin ribozyme cleavage
were 10 mM MgCl2 at 25 °C using various buffers at 25 mM
concentration depending on pH and 50 mM NaCl (pH 5−5.5 acetate,
pH 5.5−6.75 MES, pH 6.75−8 HEPES, pH 8−9 TAPS). Products of
the cleavage reaction were analyzed via denaturing PAGE and were
quantified by phosphorimaging.
Analysis of Ribozyme Kinetics and Analysis of pH Depend-
ence of Reaction Rate. Substrate cleavage was studied under single-
turnover conditions as fully described in the SI. The pH dependence
of observed cleavage rates was fitted to either a single-ionization model
incorporating a pH-independent contribution observable at low pH
(k0),
Figure 1. Hairpin ribozyme and a proposed reaction mechanism. (A)
Proposed mechanism of cleavage and ligation based on general acid−
base catalysis. This depicts the transition state; the arrows show the
flow of electrons for cleavage. (B) The sequence of the hairpin
ribozyme used here. The ribozyme comprises four strands labeled a−d.
in a manner that is consistent with their proposed roles in
general acid−base catalysis (Figure 1A).
kcleave
kobs
=
+ k0
1 + 10pK −pH
A
(1)
However, the role of nucleobase-mediated general acid−base
catalysis in the hairpin ribozyme, and particularly the role of
G8, has been controversial. This originally stems from the pH
dependence of the cleavage and ligation reactions. In contrast
to the VS ribozyme (that exhibits a bell-shape dependence of
reaction rate on pH consistent with a role for two nucleobases
of relatively low and high pKa values15), the rates of the hairpin
ribozyme reactions increase with pH to approximately
neutrality, and then remain at a plateau at higher pH
values.11,13,21 This appears to suggest that the reactions depend
on the ionization of a single group. Coupled with the results of
exogenous base rescue experiments, this led to the suggestion
that A38 acts not as a general acid, but instead by the
electrostatic stabilization of the transition state.13,22 However,
when G8 is substituted by a nucleobase of lower pKa the
cleavage reaction rate falls with pH to generate a bell-shaped
dependence,11,23,24 and it is clear that these modified hairpin
ribozymes, just like the VS ribozyme, require two groups to be
in the appropriate ionization state for activity.
or a double-ionization model:26
kcleave
+ 10pK
kobs
=
A2 −pH
A2 −pKA1
1
1 + 10pK
+ 10pH−pK
A
(2)
where kcleave is the intrinsic rate of the cleavage reaction and pKA1 and
pKA are the acid dissociation constants of two titrating functional
2
groups.
RESULTS
■
The Construction of the Hairpin Ribozyme. In this
study we have focused on the natural form of the hairpin
ribozyme, based on a four-way junction8 (Figure 1B). This was
constructed by the hybridization of four synthetic oligonucleo-
tides, including the radioactively [5′-32P]-labeled substrate
strand d in which the scissile phosphate was either unmodified
(5′-PO) or incorporated a 5′-phosphorothiolate linkage.16,25,27
Complete sequences for each strand are given in the
Supporting Information. For synthetic convenience the
substrate strand was extended 3′ to the cleavage site beyond
the six nucleotides used in previous studies.21,23 We therefore
shortened strand a in order to retain just three base pairs with
the 3′ product of cleavage, that is known to result in rapid
dissociation of the 3′ product28 and thus to eliminate the
reverse reaction (i.e., ligation). This new construct exhibited a
cleavage rate that was indistinguishable from that observed for a
hairpin ribozyme species with a 5′-extended a-strand.23 The 5′-
PS substrate strand included o-nitrobenzyl protection of the 2′-
hydroxyl of the adenosine to prevent premature activation of
5′-Phosphorothiolate (5′-PS) substitution of the scissile
phosphate provides a powerful means to examine the proposed
role of nucleobases in general acid−base catalysis, and has
previously illuminated the mechanisms of the hepatitis delta
virus25 and VS ribozymes.16 Sulfur is a far better leaving group
than oxygen and does not require protonation by a general acid.
If substitution of a particular nucleobase leads to loss of
catalytic activity because it normally (i.e., in the unmodified
case) protonates the leaving group as a general acid, then
activity should be restored by the 5′-PS substitution. By
contrast, if the nucleobase accelerates the reaction by acting as a
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the nucleophile; its synthesis is described in the Supporting
Information.
Table 1. Rates of Cleavage for Different Hairpin Ribozymes
Measured under Single-Turnover Conditions in 25 mM
HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2 at 25°C
Incorporating an Unmodified Substrate (5′-PO) or a 5′-
The Activity of a Hairpin Ribozyme with a Purine at
Position 38 Is Restored by 5′-Phosphorothiolate
Substitution in the Substrate. Substrate strand cleavage
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 phosphor-
imaging. The unmodified substrate (i.e., 5′-PO) strand
exhibited no detectable cleavage after 20 min incubation
under standard conditions (25 mM HEPES (pH 7.5), 50 mM
NaCl, 10 mM MgCl2 at 25 °C) in the absence of the ribozyme
(Figure 2A).
a
Phosphorothiolate-Linked Substrate (5′-PS)
5′-PS,
5′-PS kobs/5′-PO
ribozyme
natural
5′-PO, kobs/min−1
k
obs/min−1
kobs
0.51 0.08
5.8 0.8
11
ribozyme
b
none
u.d.
0.004 0.003
0.020 0.002
2.0 0.2
−
−
180
b
ΔB ribozyme
G8DAP
G8I
u.d.
0.011 0.002
0.02 0.02
0.17 0.03
0.17 0.003
0.025 0.002
0.5 0.1
8
A10U
0.0147 0.0002
0.014 0.003
0.00015 0.00003
12
C25U
2
A38P
3600
−
b
G8DAP/A38P
a
u.d.
0.14 0.04
Each rate constant is the average of ≥3 independent measurements.
b
u.d. denotes a rate of <10−5 min−1, i.e. essentially undetectable
activity after >40 h incubation.
Ribozymes with substitutions detrimental to activity each
cleave the 5′-PS substrate more rapidly than the 5′-PO
substrate (Figure 2A, Table 1). However, the ribozyme in
which A38 has been substituted by purine (A38P) stands out. It
is the substitution most deleterious to cleavage of the 5′-PO
substrate, yet it shows the greatest extent of restoration of
activity by the 5′-PS substitution (3600-fold). The almost
complete restoration of cleavage activity for the A38P ribozyme
in the presence of the 5′-PS linkage is consistent with the
assignment of A38 as the putative general acid in the cleavage
reaction catalyzed by the hairpin ribozyme.
The pH Dependence of Activity of the A38P
Ribozyme Is Consistent with General Acid−Base
Catalysis. The data presented above are consistent with A38
acting as a general acid to protonate the 5′-oxyanion leaving
group in cleavage, but it is necessary to examine activity as a
function of pH for this to be substantiated and to examine the
role of the putative general base. The cleavage and ligation rates
of the hairpin ribozyme are pH dependent with identical
shapes. They increase with pH until neutrality is reached and
remain constant over the measurable pH range thereafter.13,21
The proposed general acid−base catalysis mechanism for
hairpin ribozyme cleavage requires a protonated A38 and a
deprotonated G8 as active components (Figure 1). Thus, the
observed rate of cleavage (kobs) will be given by:
Figure 2. Cleavage of the 5′-PO or 5′-PS substrate by some hairpin
ribozyme variants. The substrate strand was [5′-32P]-labeled, and
reaction products were separated by electrophoresis in 20%
polyacrylamide and subjected to phosphorimaging. (A) Gel electro-
phoresis. The 5′-PO (tracks 1, 3, 5, 7 and 9) and 5′-PS (track 2, 4, 6, 8
and 10) substrates were incubated with no ribozyme (track 1, 2), the
natural hairpin ribozyme (track 3, 4), A38P (track 5, 6), A10U (7, 8)
or ΔB ribozyme where loop B was removed by complementation
(track 9, 10) for 20 min under standard conditions. (B) Reaction
progress of the cleavage reaction under standard conditions, fitted to
double exponential functions. Open circles: 5′-PS + A38P ribozyme,
filled squares: 5′-PO + natural ribozyme, filled triangles and filled
circles (inset): 5′-PO + A38P ribozyme.
kobs = kcat × fA × fB
(3)
where fA and fB are the fractions of A38 and G8 in their
protonated and deprotonated states, respectively (which can be
calculated for any pH from their assumed pKa values), and kcat
is the rate of cleavage by ribozyme with acid and base in their
required forms. Following the approach of Bevilacqua,26 in
Figure 3A we simulated the expected pH profile of the reaction
for pKa values of 6 and 10.5, corresponding to A38 and G8
respectively.13,21,29
The rise in rate at low pH is due to the deprotonation of the
guanine (i.e., the rise in fB, which occurs over the whole range
of the titration), while the formation of the plateau is due to the
deprotonation of the adenine (i.e the fall in fA) offsetting the
rise in f B. Thus the pKa measured is that of the adenine. If the
5′-PS substitution obviates the requirement for leaving group
protonation, then a general acid is no longer required for
Only a very small fraction of the corresponding 5′-PS
substrate was cleaved under these conditions. Hybridization
with strands b−d to form the hairpin ribozyme led to significant
cleavage of the 5′-PO substrate. The 5′-PS substrate strand is
cleaved faster, so that a greater extent of cleavage following the
20 min incubation was observed; this might be anticipated if
the general acid protonating the leaving group is significantly
deprotonated at pH 7.5 (see below). Graphs of reaction
progress are shown in Figure 2B, and measured rates tabulated
in Table 1.
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base cleavage mechanism with the nucleobase at position 38
acting as the general acid and a species with high pKa acting as
the general base.
Nucleotide Substitutions of G8 in the Hairpin
Ribozyme Influence the pH Profile for Cleavage of the
5′-PS Substituted Substrate Strand. The log−linear
increase in cleavage rate with pH for the 5′-PS substrate
catalyzed by the A38P ribozyme is consistent with general base
catalysis by G8, but to test this conclusion we have substituted
it with a different nucleotide with a markedly lower pKa. This
should result in a predictable change in the pH-activity profile
(Figure 4A).
Figure 3. The pH dependence of cleavage rates for the 5′-PO and 5′-
PS substrate with various ribozyme constructs. (A) Simulation of the
reaction rate as a function of pH for a general acid of pKa = 6 and a
general base of pKa = 10.5. The fractions of the protonated acid (fA −
red dotted line), unprotonated base (f B − green solid line) and fA × f B
(dashed line) are plotted as a function of pH. In all simulations, the
shaded regions lie outside the experimentally accessible range of pH;
note that the reduction in fA × f B at high pH falls outside the
observable region. If general acid catalysis is not required, the pH
dependence should simply follow f B. (B) Experimental cleavage rates
for the 5′-PO and 5′-PS substrate with various ribozyme constructs are
plotted on a logarithmic scale as a function of pH. Filled triangles, 5′-
PS substrate + natural-sequence ribozyme; filled circles, 5′-PS
substrate + A38P ribozyme; filled diamonds, 5′-PS substrate + ΔB
ribozyme (lacking loop B); open circles, 5′-PO substrate + A38P
ribozyme. The data for the 5′-PS substrate + A38P ribozyme have
been fitted to a single ionization model with additional pH-
independent component as discussed in the text, yielding an apparent
pKa of 8.5 0.2.
Figure 4. The pH dependence of ribozyme cleavage rates for the 5′-
PO and 5′-PS substrate where G8 has been substituted by
diaminopurine. (A) Simulation of reaction rate as a function of pH
for an acid of pKa = 6 and a base of pKa = 7 (corresponding to DAP).
The fractions of the protonated acid (fA − red dotted line),
unprotonated base (f B − green solid line) and fA × f B (dashed line)
are plotted as a function of pH. Note that the pH dependence should
now be bell-shaped within the observable range. In addition, the
fraction of unprotonated base (f B) for a pKa = 10.5 is also plotted to
simulate ribozyme with an unmodified G8 (solid line). (B)
Experimental cleavage rates (plotted on a logarithmic scale) for the
5′-PO and 5′-PS substrates + G8DAP hairpin ribozyme. Filled circles,
5′-PS substrate + A38P, G8DAP ribozyme (data fitted to a single
ionization model with k0 = 0, yielding an apparent pKa of 5.5 0.2);
open squares, 5′-PO substrate + G8DAP ribozyme (data fitted to a
double ionization model, pKa1 = 5.1 0.1, pKa2 = 7.3 0.1). The fit to
the data for the 5′PS substrate + A38P ribozyme has been plotted for
comparison.
catalysis and the pH profile of activity would be expected to
reflect only the extent of deprotonation of the general base (i.e.,
fB, not the product fA × f B). Thus, the rate of cleavage of the 5′-
PS substrate would be expected to increase in a log−linear
manner over the observable pH range, diverging from the pH
profile for cleavage of the 5′-PO substrate at the pKa of the
general acid. Experimentally the observed activity is almost
constant over the range of pH investigated (Figure 3B). Thus,
either the cleavage of the 5′-PS substrate is pH independent,
and thus not subject to general base catalysis by G8, or the
cleavage reaction is not rate-limiting under these conditions.
In order to resolve this ambiguity we investigated the pH
dependence of activity of the A38P ribozyme (Figure 3B),
where the rates of cleavage are lower. For the unmodified 5′ PO
substrate the cleavage rate remains constant at approximately
For this purpose we investigated the activity of a modified
hairpin ribozyme with 2,6-diaminopurine (pKa = 5.1) at
position 8 (G8DAP). This substitution leads to a bell-shaped
pH profile with a center near pH = 6 for the cleavage of the 5′-
PO substrate (Figure 4B), because the saturation of the
deprotonation of the base is now within the observable pH
range. A similar pH dependence has previously been found for
the minimal form of the hairpin ribozyme with the same
G8DAP substitution.11 Fitting the curve to a double-ionization
model gives two apparent pKa values of 5.1 0.1 and 7.3 0.1,
possibly corresponding to A38 and G8DAP, respectively.
In order to determine the effect of a G8DAP substitution on
the pH profile for cleavage of the 5′-PS substituted substrate,
we investigated the activity of a ribozyme with both A38P and
G8DAP substitutions. This ribozyme has no detectable activity
toward the 5′-PO substrate, while the pH profile for the
cleavage of the 5′-PS substrate rises up to pH = 6.5 and remains
constant at higher pH (Figure 4B). The apparent pKa value of
5.5 0.2 is consistent with the deprotonation of 2,6-DAP. The
shift in apparent pKa on substitution of G8 with nucleobases of
lower pKa supports the proposition that the nucleobase at
k
obs = 10−4 min−1 over the measurable pH range. However, for
the 5′-PS substrate, the cleavage rate rises monotonically with
increasing pH and is approximately log−linear over the range of
pH 6−8. However, the deviation from linearity below pH 6 is
not consistent with general base catalysis alone. This deviation
could be due to the presence of an additional pH-independent
reaction pathway that contributes significantly to catalysis at
low pH. For example, this might involve removal of a proton
from the 2′-OH nucleophile by the pro-R nonbridging oxygen
of the scissile phosphate as proposed on the basis of
computational modeling of the reaction pathway.30 Alter-
natively protonation of a nucleobase close to the cleavage site
such as A10 might contribute to catalysis.31 This could stabilize
the transition state and result in the rate enhancement observed
at low pH. Fitting the data to a single-ionization model
incorporating a low-pH channel to catalysis yields an apparent
pKa of 8.5 0.2 for the general base. Thus, allowing for an
additional contribution to catalysis observable at low pH, the
data for the A38P ribozyme are consistent with a general acid−
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position 8 is acting as a general base in hairpin ribozyme
catalysis.
apparent pKa of ribozyme cleavage,33 and nucleotide analogue
interference mapping revealed a strong interference at position
38 from adenosine analogues of low pKa,31 consistent with
ionization of N1 influencing ribozyme activity. Finally, A38 has
an elevated ground-state microscopic pKa of 5.46 in a
precatalytic conformation of the hairpin ribozyme, as measured
by Raman crystallography,34 which correlates well with the
macroscopic, apparent pKa of hairpin ribozyme activity. We
consider that these results, together with our new data,
demonstrate that A38 protonates the 5′-O leaving group in
the cleavage reaction of the hairpin ribozyme.
Activity of Hairpin Ribozyme Lacking Loop B on
Substrate Carrying a 5′-PS Substitution. If the main
function of loop B is to deliver A38 to act as the general acid for
the cleavage reaction, it might be anticipated that ribozyme with
loop A alone could retain some level of activity when the
substrate carries the 5′-PS substitution. We therefore
investigated a hairpin ribozyme variant in which only loop A
is present by inclusion of an alternative b strand that
complements loop B while preserving the four-way junction
(termed ΔB ribozyme). This ΔB ribozyme exhibits negligible
cleavage activity on the 5′-PO substrate (Table 1), confirming
that loop B (which includes A38) is absolutely required for
catalysis. By contrast, the presence of ΔB ribozyme produces a
5-fold greater rate of cleavage of the 5′-PS substrate compared
to the 5′-PS substrate alone (Table 1). However, the rate is
significantly lower than that observed for the natural and A38P
ribozymes. The reaction rate exhibits log−linear dependence
on pH, almost parallel to the A38P hairpin ribozyme but with
lower absolute rates (Figure 3B). This is consistent with G8
acting as general base in both cases.
The cleavage rate of the 5′-PS substrate by the A38P hairpin
ribozyme exhibits the log−linear dependence on pH expected if
the nucleophile is activated by a general base of high pKa. In
contrast, the activity of the natural hairpin ribozyme toward the
5′-PS substrate is almost constant over the pH range
investigated. In this case either the cleavage of the 5′-PS
substrate is pH independent, and thus not subject to general
base catalysis by G8, or the cleavage reaction is not rate limiting
under these conditions. A pH-independent activation of the
nucleophile is not consistent with the data for the natural
ribozyme (see below) but cannot be excluded for cleavage of
the 5′-PS substrate. However, we favor the possibility that a
conformational change is limiting the activity of the ribozyme.
We have previously shown that the rate of oscillation by the
four-way junction between parallel and antiparallel states is
∼100 min−1 in the standard buffer used here.21,35 As the loop−
loop interaction necessary for ribozyme activity occurs with the
junction in the antiparallel state, the dynamics of the junction
sets an upper limit on the activity of the ribozyme that is only 1
order of magnitude greater than the rates observed for cleavage
of the 5′-PS substrate by the natural ribozyme. The extensive
remodeling of both loops A and B to form the active ribozyme
(revealed by comparison of the NMR structures of the isolated
loops36,37 and the crystal structure of the ribozyme17)
presumably occurs subsequently to initial contact and may
further limit the rate of formation of the active ribozyme.38 The
observation that the natural ribozyme and a ribozyme with a
G8DAP substitution exhibit almost identical maximum rates of
cleavage of the 5′-PS substrate yet very different rates of
cleavage of the 5′-PO substrate (Table 1) also suggests that
conformational changes may limit the activity of these
ribozymes.
The importance of remodeling loop A to obtain an active
structure is emphasized by our data for the ΔB ribozyme
(Table 1). These results indicate that cleavage of the 5′-PS
substrate can proceed in the absence of loop B and that in this
situation a species of high pKa is activating the nucleophile.
However, the lower rate compared to that of the A38P
ribozyme shows that the interaction between loop A and loop B
is important in generating the catalytically competent
conformation. This is seen also in the data for the A10U and
C25U substitutions, which weaken the loop−loop interaction
through disruption of the ribose zipper and a critical G+1:C25
base pair, respectively.11,15,36 The limited suppression of these
defects by the 5′-PS substrate is consistent with an impairment
in loop A remodeling.
DISCUSSION
■
5′-Phosphorothiolate substitution of the scissile phosphate was
used to address the functions of A38 and G8 in hairpin
ribozyme catalysis, and in particular their proposed roles as
general acid and general base, respectively. 5′-PS substitution
obviates the need for protonation of the leaving group,
rendering the action of the general acid unnecessary. The
effects of modifications to the ribozyme that decrease the
activity of the general acid are potentially suppressed by the 5′-
PS substitution, whereas other modifications should affect 5′-
PO and 5′-PS substrates to a similar extent. Thus, the identity
of the general acid can be established, and the function of the
general base can be studied in isolation. The effects of
nucleobase substitutions to the hairpin ribozyme and the pH
dependence of their rates are consistent with guanine at
position 8 acting as general base and adenine at position 38
acting as general acid to provide a significant contribution to
catalysis in the hairpin ribozyme.
The activity of an impaired ribozyme containing a purine
nucleobase at position 38, which generates negligible cleavage
of a 5′-PO substrate, was restored to the level of the natural
ribozyme by 5′-PS substitution at the cleavage site. All other
substitutions were suppressed by the 5′-PS modification to a
much smaller extent (Table 1). This is consistent with A38
acting as a general acid in protonating the leaving group. If the
contribution of A38 to catalysis is solely, or indeed
substantially, through electrostatic stabilization of the transition
state, as has been proposed,14 an effect of this magnitude would
not be expected.
This conclusion is in accord with previous evidence, reviewed
in Wilson and Lilley.29 A ribozyme with N1-deazaadenine at
position 38 has no measurable activity although it folds into an
apparently unperturbed active structure.32 Replacement of A38
with isoguanine, which has the N6 exocyclic amine of adenine,
but a pKa similar to that of guanine, gives a ribozyme with
negligible activity at neutral pH. However, at high pH its
cleavage activity is as great as that of the native ribozyme,14
consistent with general acid−base catalysis by two nucleobases
of high pKa. Replacement of A38 with 8-azaadenine, which has
a lower pKa, resulted in a corresponding decrease in the
The pH dependence of cleavage of the 5′-PS substrate by the
A38P hairpin ribozyme is consistent with the action of a general
base of high pKa. We have previously argued that the
substitution of G8 with nucleobases of lower pKa provides
direct support for G8 fulfilling this role.29 The bell-shaped pH
dependence and apparent pKa values observed on substitution
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of G8 with diaminopurine, 2-aminopurine,11 imidazole,23 or 8-
azaguanine24 are consistent with the nucleobase at position 8
acting in concert with A38 in general acid−base catalysis.
Similarly, rescue of an abasic lesion at position 8 by exogenous
cytosine or 2-aminopyridine13 is consistent with the hypothesis
that the exogenous base is acting as a general base during
cleavage. Furthermore, the structure of the cleaved ribozyme
has G8 positioned to act as a general acid in the ligation
reaction, with N1 donating a hydrogen bond to the O2′ leaving
group.20 If it so acts, then by the principle of microscopic
reversibility it should act as a general base during cleavage. To
test if G8 does fulfill this role we investigated the cleavage of
the 5′-PS substrate by a ribozyme with 2,6-diaminopurine at
position 8, retaining the A38P substitution. The pH depend-
ence of activity reflects the deprotonation of a base with a much
lower apparent pKa, consistent with that of 2,6-diaminopurine.
Since the pH profile is expected to correspond to the
protonation state of the general base in this experiment, the
lower apparent pKa supports the proposition that the
nucleobase at position 8 is directly involved as general base
in the catalytic mechanism of hairpin ribozyme cleavage.
It is interesting to note that the apparent pKa values of
nucleobases at position 8 measured in the context of the A38P
and 5′-PS substitutions (8.5 for guanine and 5.5 for 2,6-
diaminopurine) are substantially lower than those inferred from
the pH profiles for ribozymes with A38 and a natural substrate
(10.6 for guanine,29 7.3 for 2,6-diaminopurine). This might
reflect limits to activity that result from conformational change.
Alternatively the loss of hydrogen-bonding potential due to the
A38P and 5′-PS substitutions may result in a change in the local
environment of the nucleobase at position 8.
Figure 5. General acid−base catalysis yields equivalent pH depend-
ence for cleavage and ligation. The roles of general acid and base will
be exchanged between cleavage and ligation reactions. However, the
rates of reaction are proportional to fA × fB, which have the same
shape for both forward and reverse reactions, although the magnitudes
are very different. (A) Simulation of reaction rate as a function of pH
for an acid of pKa = 6 and a base of pKa = 10.5, corresponding to the
proposed mechanism for cleavage. The upper panel plots the fractions
of the protonated acid (fA) and unprotonated base (fB) in red dotted
and green solid lines, respectively, with fA × f B shown in the lower
panel. (B) Simulation of the reaction rate as a function of pH for an
acid with pKa = 10.5 and a base with pKa = 6 corresponding to the
proposed mechanism for ligation, following the presentation in A.
Note that the pH dependence of f B and the product fA × fB are
experimentally indistinguishable for the ligation reaction, in contrast to
the cleavage reaction shown in A.
We further note that the natural ribozyme exhibits
significantly faster cleavage of the 5′-PS substrate than of the
5′-PO substrate, even at pH = 5, where A38 is expected to be
almost fully protonated. This implies a corresponding enhance-
ment in kcat. This may be due to a greater intrinsic reactivity
because sulfur is a better leaving group, although a similar
enhancement in activity at low pH was not observed for the
HDV25 or VS ribozymes.16 Alternatively, the increase in rate
may be due to a greater fraction of ribozyme molecules having
an active conformation, a possibility suggested by the crystal
structure of an inactive hairpin ribozyme with a 2′-O-methyl
substitution of the nucleophile.17 Despite G8 being in
proximity to the nucleophile and a near-optimal in-line
orientation of nucleophile and leaving group, an unmodified
ribozyme having the same conformation as the crystal structure
would not be reactive since A38 lies some distance from the
leaving group. Yet in the context of the 5′-PS substitution,
where protonation of the leaving group by A38 is not required,
such a structure should be active and may account for the
enhanced rate.
General acid−base catalysis by A38 and G8 is fully consistent
with the data, but can other mechanisms provide an alternative
explanation? The equivalent pH dependence observed for
cleavage and ligation reactions13,21 significantly constrains the
possible mechanisms. Equivalent pH profiles are readily
explained by the ionization of two groups (Figure 5).
The curves of the product fA × f B as a function of pH have an
identical shape for the cleavage and ligation reactions, and while
the magnitude of fA × fB is much higher for ligation in the case
illustrated, this will be offset by a lower kcat due to both
nucleobases being in a less reactive, uncharged state.26 In
contrast, ionization of a single group that participates in proton
transfer would yield inverted pH profiles for cleavage and
ligation. In principle, the pH dependence of the ligation
reaction of the hairpin ribozyme is consistent with a single
group with pKa ≈ 6, such as A38, acting as a general base in a
stepwise mechanism where formation of the bond between the
5′-O and P is rate limiting. In this case the rate of reaction
would be proportional to the magnitude of f B (Figure 5B), but
by the principle of microscopic reversibility the cleavage
reaction rate would be proportional to fA for the same group
(Figure 5A), and thus an inverted profile would be expected in
this case; this is not consistent with observation. However,
ionization of a single group is consistent with equivalent
profiles for cleavage and ligation if the group is involved in
electrostatic transition-state stabilization or indirectly influences
activity, for example by affecting a rate-limiting conformational
change.
Accepting that A38 protonates the leaving group in the
cleavage reaction, equivalent pH dependence of cleavage and
ligation reactions is only consistent either with A38 acting
together with a second ionizable group, or with the reaction not
being limited by chemistry. Yet in the latter scenario, the state
of protonation of A38 should not affect the pH dependence of
the reaction, whereas there is good evidence that titration of
A38 does determine the pH dependence of the reaction, as
discussed above. We conclude that A38 acting in concert with a
second ionizable group is the only mechanism that is consistent
with the data.
The identity of the functional group activating the
nucleophile in the cleavage reaction has been the subject of
considerable investigation. A shuttle mechanism in which A38
removes a proton from the nucleophile and donates it to the
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Journal of the American Chemical Society
Article
leaving group has been proposed,39 but this mechanism is
necessarily stepwise and would be expected to yield inverted
pH profiles for cleavage and ligation. Both the pro-R and pro-S
nonbridging oxygens have been considered,30,39,40 but the
nonbridging oxygens are fully deprotonated over the observable
pH range. Thus, the dependence of cleavage activity on pH
would be expected to parallel the fractional protonation of the
acid (fA in Figure 5A), which is opposite to what is observed.
However, removal of a proton from the nucleophile by a
nonbridging oxygen could, in principle, explain the data for the
cleavage of the 5′-PS substrate by the natural ribozyme, but not
those for the A38P ribozyme. It may also explain the deviation
from linearity below pH 6 observed in the cleavage of the 5′-PS
substrate by the A38P ribozyme.
All the available data support a high pKa for the second
ionizable group. Of the nucleobases with a high pKa only G8
lies close to the O2′ in the crystal structure. The hairpin
ribozyme functions efficiently in the absence of divalent cations
so a magnesium ion-bound hydroxide ion is not necessary for
activity.3,41−43 Although divalent cations may contribute to
catalysis under some conditions, they cannot account for the
low pKa observed with 2,6-diaminpurine at position 8.
Bevilacqua26 has argued that water lacks the necessary catalytic
power, but it remains a possibility that for the natural ribozyme,
where the pKa of the general base is outside of the observable
range, a water molecule activated by a specific hydrogen-
bonding network removes a proton from the nucleophile
instead of G8. This role for water is plausible so long as the
upper pKa is not observable but is difficult to reconcile with the
lowered pKa for the general base observed for ribozymes with
substitutions at position 8 (such as G8DAP) that exhibit a bell-
shaped pH profile. It seems probable that these ribozymes use a
species other than water to activate the nucleophile, and in each
case the observed pKa for the general base is consistent with the
nucleobase substituted for G8 taking this role. In the absence of
evidence to the contrary we strongly favor the simplest and
most consistent hypothesis that the pH dependence of hairpin
ribozyme activity arises from general acid−base catalysis by A38
and G8.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Descriptions of the chemical synthesis of A[2′-O-(o-nitro-
benzyl)]-psG and the ligation of the substrate and construction
of the hairpin ribozyme. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
d.m.j.lilley@dundee.ac.uk
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Scott McPhee for oligoribonucleotide synthesis, the
Deutsche Forschungsgemeinschaft (S.K.S. research fellowship),
and Cancer Research UK (D.M.J.L. lab) and NIH (Grant:
R01AI081987 to J.A.P.) for financial support.
REFERENCES
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