(Received for publication, September 27, 1995)
From the
The hairpin ribozyme is a 50-nucleotide RNA enzyme of unknown
three-dimensional structure. Here, we demonstrate that interdomain
interactions are required for catalytic function by reconstitution of
activity following separation of an essential, independently folding
domain (loop B) from the substrate binding strand at a helical
junction. The resulting construct relies on long range tertiary
contacts for catalysis. For this work, we used an optimized ribozyme
and substrate, which included sequence changes to minimize the
formation of nonproductive conformational isomers. Kinetic analysis was
carried out using both single and multiple turnover methods and shows
that the catalytic efficiency (k/K
) of the
reconstituted ribozyme is 10
-fold lower than that of the
intact ribozyme. The decrease in k
/K
results
entirely from a 10
-fold increase in the apparent K
, whereas the k
parameter is essentially unchanged. Therefore, cleavage chemistry
appears to be unimpaired, but the reaction is limited by the productive
assembly of the two domains. Our results strongly support a previously
proposed model in which the catalytic topology of the ribozyme contains
a bend at a helical junction.
The hairpin ribozyme is derived from the satellite RNA of the
tobacco ringspot virus and catalyzes the reversible, site-specific
cleavage of RNA substrates, generating 5`-OH and 2`,3`-cyclic phosphate
termini(1, 2, 3) . Nucleotides and functional
groups that are essential for catalytic function have been identified
by several methods, including mutagenesis(4, 5) , in vitro selection(6, 7, 8, 9) , and the
introduction of modified
nucleotides(10, 11, 12) . Interestingly,
essential elements of the ribozyme are concentrated within a large
asymmetrical internal loop (loop B) and a smaller symmetrical loop
formed through binding of the substrate to the substrate binding strand
(SBS) ()(see Fig. 1). The finding that many of the
essential determinants reside within loop B strongly suggests that this
portion of the molecule interacts with the rest of the
ribozyme-substrate complex during catalysis. Linker insertion (13, 14) and chemical modification studies (15) of the helix 2-3 junction indicate that helix 2 is
not coaxially stacked upon helix 3, i.e. the
ribozyme-substrate complex has a bend between helices 2 and 3. Such a
bend would presumably be required to bring essential elements in loop B
proximal to the cleavage site.
Figure 1:
Domain separation strategy and
reconstitution of catalysis. A, secondary structure of the
optimized hairpin ribozyme ( (30) and this work). Substrate
cleavage site is indicated with an arrow. The loop B domain
was separated at the junction between helices 2 and 3, yielding a
three-piece ribozyme consisting of a duplex between substrate and
substrate binding strand (SSBS) together with the loop B domain. B, reconstitution of catalysis. Reactions were for 30 min at
37 °C in a buffer containing 50 mM Tris
Cl, pH 7.5,
100 mM MgCl
. S, 1 nM 5`
end-labeled substrate only; S + SBS, 1 nM substrate in the presence of excess (15 µM) SBS; S + LB, substrate in the presence of 10
µM loop B domain; S + SBS + LB, reconstituted reaction with 1 nM substrate, 15
µM SBS, 10 µM loop B domain; S + HpRz, substrate incubated with 60 nM
intact, optimized hairpin ribozyme as shown in A, left. 5`P, 5` cleavage
product.
Local tertiary interactions within
loop B have been studied by UV cross-linking (16) and chemical
modification analysis(15) . These studies indicate that a
photosensitive structural motif occupies the segment of loop B that is
proximal to helix 3. This portion of the ribozyme is strikingly similar
to the conserved central domain of viroids(17) , to loop E of
eukaryotic 5 S rRNA, and to the sarcin-ricin loop of 26 S rRNA. The
latter two structures have been analyzed using NMR
methods(18, 19, 20) . These structures are
comprised of non-canonical GA, A
U, and A
A base pairs
across the loop and result in a helical conformation with accessible
major and minor grooves. The reason a common structural motif is found
in several functionally distinct RNAs is not yet clear, but it has been
proposed that the structure may serve as a common ``docking
module'' for RNA-RNA or RNA-protein
interactions(20, 21) . In the hairpin ribozyme, the
cross-linkable loop B structure is clearly essential for catalytic
function. Furthermore, results of chemical modification experiments
suggest that magnesium-dependent higher order structure is superimposed
on the photoreactive motif(15) .
Cross-linking and chemical
modification results indicate that the loop B domain of the hairpin
ribozyme, defined as helix 3, loop B, and helix 4, folds into its
correct structure independently of substrate and SBS (15, 16) . We took advantage of this property to
search for functional interactions between loop B and a duplex
consisting of substrate bound to the SBS (SSBS). Using this
strategy, we have been able to reconstitute the cleavage reaction. The
interdomain interactions demonstrated by this reconstitution are likely
to be tertiary contacts, since little potential for formation of
canonical base pairs exists between the domains and no phylogenetic
evidence for interdomain secondary structure could be obtained by
analyzing a large number of sequence
variants(6, 7, 9) .
The naturally occurring substrate sequence for the hairpin
ribozyme (UGACAGUCCUGUUU) is conformationally heterogeneous and
migrates as multiple conformations on native polyacrylamide gels (28) . (
)In general, it was found that
conformationally heterogeneous substrates and ribozymes showed
unsatisfactory kinetic behavior. To design a well behaved substrate and
SBS, we varied sequences in such a manner that the conformational
heterogeneity is minimized while maintaining sequences essential for
catalytic function (Fig. 1). Conformational homogeneity of the
new substrate and SBS over a concentration range of 1 nM to 40
µM was confirmed by native gel electrophoresis (data not
shown). We utilized a stabilized loop B domain in which helix 4 was
extended by the addition of seven base pairs and a stable GUAA
tetraloop (29) and which contained a rate-enhancing U39C
mutation(7, 9) . These modifications each
approximately double the catalytic efficiencies of intact hairpin
ribozymes and produce a loop B domain that gives only one detectable
conformation, which is the active and cross-linkable
structure(16, 30) .
To reconstitute a cleavage
reaction between separated loop A and B domains, loop A duplexes were
preformed by annealing substrate to its cognate SBS. Cleavage activity
was observed only when the loop B domain was incubated with the
SSBS duplex (Fig. 1B). No reaction could be
detected in the absence of the loop B domain or in the absence of the
substrate binding sequence, even upon extended incubation times. The
reaction occurs at the normal site, as evidenced by product mobility,
and no inaccuracy of cleavage is detectable. These results clearly
demonstrate a functional interaction between the two domains of the
ribozyme-substrate complex.
The background rate of hydrolysis of
substrate RNA was measured in the absence of ribozyme, under buffer and
temperature conditions identical to those used in the Fig. 1reconstitution experiment (data not shown). The measured
rate was approximately 10 min
,
which is similar to previously reported values for RNA
hydrolysis(31) . Similar background rates were observed in the
presence of a 100-1000-fold excess of SBS, indicating that formation of
the loop A domain does not result in an increased lability of the
scissile bond. In these latter experiments, 1 nM 5`
end-labeled substrate was incubated with 100 and 1000 nM SBS
for up to 48 h. Similarly, no increased lability of the scissile bond
was observed with substrate incubated in the presence of loop B (5
µM) with no SBS present.
Kinetic analysis of the
bisected ribozyme construct provides useful insights into interactions
between the domains. To ensure that substrates remained bound to
substrate binding strands in the form of SSBS duplexes, we first
determined the apparent K
for the SBS in the
ternary complex (Fig. 2). The apparent K
was observed to be approximately 70 nM for the SBS, and
saturation was observed in all cases where SBS concentration was
5
µM. In these experiments, K
was
observed to be independent of loop B concentration. To maintain the
S
SBS duplex in subsequent experiments, we chose to use an excess
of SBS relative to S, such that SBS concentrations were always 15
µM or greater.
Figure 2:
Apparent K of the SBS in the reconstituted reaction. 1 nM 5`
end-labeled substrate was bound to varying concentrations of SBS, from
0.01 to 100 µM, and the initial velocities of the
reactions at each SBS concentration were determined by single turnover
kinetics as described under ``Experimental Procedures.'' The
experiment was performed with three different concentrations of the
loop B domain as indicated, and the initial velocities were normalized
to the maximum velocity obtained with 75 µM loop B. An
apparent K
value of approximately 70
nM was obtained for all three loop B domain concentrations. In
these experiments, K
describes
interactions between S and SBS.
We used both pre-steady state (single
turnover) and steady state (multiple turnover) kinetic analysis to
describe the reconstituted reaction. Single turnover reactions were
performed by using a trace amount of radioactive substrate prebound to
SBS and increasing amounts of the loop B domain. Loop B domain was
always in significant excess over substrate. The reaction could not be
fully saturated even when the concentration of loop B was increased to
400 µM (Fig. 3, A-C). The observed
reaction velocities at 400 µM loop B are within
2-3-fold of the maximum velocity of the corresponding version of
the intact ribozyme. First order rate constants obtained at eight
different concentrations of loop B were plotted, and the resulting
curve was fitted by non-linear least squares regression analysis to the
Michaelis-Menten equation, yielding a k of 0.53
min
and an observed K
of 270
µM (Fig. 3C and Table 1). The value
of k
is essentially unchanged relative to that
of the intact ribozyme, while K
for the
reconstituted reaction is increased by a factor of 10
.
These results indicate that the reconstituted reaction is likely to be
limited only by the productive association between the two ribozyme
loop domains.
Figure 3:
Kinetic analysis of the reconstituted
reaction. A, representative autoradiogram of a time course of
substrate cleavage in the presence of saturating (15 µM)
concentrations of SBS, 1 nM 5` end-labeled substrate, and 200
µM loop B domain. S, substrate; 5`P, 5`
cleavage product. B, kinetics of substrate cleavage in the
reconstituted reactions in the presence of excess loop B domain
(2-400 µM). C, initial reaction rates (k) for each time course experiment are plotted
as a function of the concentration of the loop B domain (2-400
µM). D, multiple turnover kinetic analysis. Loop
B domain (100 nM) was reacted with varying concentrations of
S
SBS duplex as described under ``Experimental
Procedures.'' Results from two independent experiments are
shown.
Multiple turnover kinetics were performed by
incubating a small amount of the loop B domain in the presence of a
large excess of the SSBS duplex and monitoring initial reaction
velocities. As in the pre-steady state analysis, we observe that the
reaction requires very high concentrations of substrate to achieve
maximum velocity. We find that the loop B domain is capable of cleaving
multiple S
SBS duplexes, with a turnover rate (k
) of 0.13 min
(Fig. 3D, Table 1). This value is about
4-fold lower than the k
value measured by single
turnover kinetics. Therefore, it appears that reactions in the presence
of a large excess (200-300 µM) of S
SBS are
slightly less efficient than reactions with a corresponding excess of
the loop B domain. This difference may be due to inhibition by high
concentrations of S
SBS (for example, RNA aggregation or
inhibition by excess substrate or SBS) or to a change in the
rate-limiting step. The steady state analysis of the reconstituted
reaction indicates that the loop B domain can cleave multiple
S
SBS complexes, but reaction efficiency is limited by a
10
-fold increase in K
.
Reconstitution of activity based on tertiary interactions has been
shown for large group I and group II
ribozymes(26, 27, 32, 33, 34, 35) .
Feldstein and Bruening (13) showed that a trans-ribozyme
restored self-ligation activity to a conformationally constrained
construct that had little or no intramolecular activity. This work
showed that intermolecular interactions could enhance hairpin ribozyme
activity but did not define the mode of interaction between the two
ribozymes. The interdomain tertiary interactions in the hairpin
ribozyme are surprisingly weak in comparison with those observed in the
larger ribozymes. For example, domain V of the self-splicing ai5
group II intron binds to the intron through tertiary interactions and
has a K
of 300 nM(26) . In
contrast, our observed value of K
for the hairpin
ribozyme domains (60-270 µM) correlates more closely
with that of the interaction between guanosine substrate and the Tetrahymena group I intron, where a K
of
320 µM is observed (36, 37) . Although we
have not demonstrated that our K
corresponds to a
dissociation constant, the notion of weak interdomain interactions is
supported by direct analysis of complex formation on nondenaturing
gels. (
)In these experiments, formation of the S
SBS
loop B ternary complex could not be detected, while S
SBS and
S
ribozyme complexes were readily identified. Therefore, it
appears that the interdomain tertiary interactions in the hairpin
ribozyme may be limited to a small number of contacts. Additionally, it
is likely that an entropic penalty accompanies domain separation,
giving rise to high apparent K
values.
In light
of the apparently high K value for interdomain
interactions in the hairpin ribozyme, the covalent linkage between the
domains in the intact ribozyme may serve to increase greatly the local
effective concentrations of the domains. Additionally, it is possible
that the phosphodiester linkage partly constrains the orientation of
the two domains, such that the frequency of productive interactions may
be increased. It is interesting to note that the interdomain
interactions described here require a strong bend at a helical junction
in the intact ribozyme; this is unusual because coaxial stacking is a
common feature of RNA structure and is energetically
favorable(38) . We expect that this reconstituted reaction will
be useful in the identification of specific interdomain interactions
and for analysis of the catalytic mechanism in the hairpin ribozyme
system.