The active form of the hairpin ribozyme is brought about by the interaction of two formally unpaired loops. In a natural molecule, these are present on two adjacent arms of a four-way junction. Although activity can be obtained in molecules lacking this junction, the junction is important in the promotion of the folded state of the ribozyme under physiological conditions, at a rate that is faster than the chemical reaction. Single-molecule fluorescence resonance energy transfer studies show that the junction introduces a discrete intermediate into the folding process, which repeatedly juxtaposes the two loops and thus promotes their docking. Using single-molecule enzymology, the cleavage and ligation rates have been measured directly. The pH dependence of the rates is consistent with a role for nucleobases acting in general acid–base catalysis.
- acid–base catalysis
- fluorescence resonance energy transfer (FRET)
- hairpin ribozyme
- RNA catalysis
- single-molecule spectroscopy
The hairpin ribozyme
The hairpin ribozyme is a member of the class of nucleolytic ribozymes that are involved in the processing of RNA molecules generated in the replication cycles of various species . These RNA molecules bring about a site-specific cleavage of the RNA backbone by means of a transesterification reaction in which the 2′-oxygen attacks the 3′-phosphorus, generating a cyclic 2′,3′-phosphate with the rupture of the bond at the 5′-oxygen atom. In general, these ribozymes can also perform the reverse, a ligation reaction, in which the 5′-oxygen is the nucleophile that attacks the cyclic phosphate.
In its natural context of the (−) strand of the tobacco ringspot virus satellite RNA, the hairpin ribozyme comprises two major structural elements. These are a four-way helical junction and two internal loops carried by the adjacent A and B arms of the junction (Figure 1). Intimate interaction between the two loops is essential for the ribozyme activity, leading to approx. 105 fold increase in the rates of site-specific cleavage or ligation reactions. Although the minimal form without arms C and D can still catalyse the cleavage reaction , its folding requires three orders of magnitude higher Mg2+ concentration  and the internal equilibrium between cleavage and ligation is shifted when compared with the natural form .
Ensemble FRET (fluorescence resonance energy transfer) studies have shown that neighbouring helical arms are co-axially stacked in pairs, A on D and B on C, with an antiparallel orientation of the continuous strands in the folded ribozyme . Thus the two loops are brought to close proximity at sub-mM Mg2+ concentrations, and extensive contacts between them have been identified in crystallographic studies of the junction form of the ribozyme [6,7].
The importance of the junction in the folding of the hairpin ribozyme
We have explored the role of the four-way junction in the folding of the hairpin ribozyme by means of FRET studies of single ribozyme molecules . Ribozymes were assembled by hybridization of four RNA strands. Donor (Cy3) and acceptor (Cy5) fluorophores were attached to the 5′-termini of the A and B arms respectively. The Eapp (apparent FRET efficiency) will be dependent on the relative proximity of the two arms, i.e. on the interaction between the loops. The molecules were attached to the glass or quartz surface through a biotin molecule conjugated to the 5′-terminus of the C arm. We have also generated the corresponding simple junction (i.e. lacking the unpaired loops) by altering the sequence of the ‘a’ strand.
Single-molecule time records of the hairpin ribozyme and its simple junction as a function of Mg2+ ion concentration are shown in Figure 2. Both exhibit fluctuations between states of high and low Eapp. The high-FRET state for the complete ribozyme has an Eapp∼0.9, whereas that for the junction has an Eapp∼0.5. The ribozyme becomes increasingly stabilized in the high-FRET state as the concentration of Mg2+ is increased; we assign this to the state in which the loops are docked together as seen in the crystal . In contrast, the simple junctions revealed a different dependence on Mg2+ ion concentration: below 20 mM, the relative fractions of high- and low-FRET states were constant, but the rate of interconversion increased with a decreased Mg2+ concentration. At concentrations below 1 mM Mg2+, the fluctuations of the simple junction were too fast for clear resolution and a cross-correlation analysis was used to determine their rates. It was found that the donor and acceptor intensities remained anti-correlated at sub-mM Mg2+ concentrations, with rates in the low-ms range (e.g. τ=8 ms in 0.5 mM Mg2+).
The same cross-correlation analysis was applied to the Eapp data for the low-FRET state of the complete ribozyme, whereupon it was found that a similar rapid fluctuation between two states occurred, with interconversion rates that were within a factor of two of those for the simple junction. Data for the low-FRET state of a single ribozyme molecule, binned at 3 ms, could be decomposed into two Gaussian distributions, centred at Eapp=0.15 and 0.4, corresponding closely to the two states of the simple junction. We conclude that the undocked ribozyme fluctuates between two states (termed UD and UP, where UD is the lowest-FRET state and UP is the intermediate-FRET state) very similar to the simple junction. This suggests that the complete ribozyme exists in three possible states (UD and UP and F, where F is the fully docked state), illustrated in Figure 3. Direct evidence for a three-state system was obtained for a sequence variant of the ribozyme, in which C25 was replaced by uridine. Time traces clearly show that the molecule exists in three states, and that the intermediate-FRET state UP is an obligate state for transitions from either the high- or low-FRET states (Figure 3).
The three-state folding suggests how the junction increases the efficiency of ribozyme folding. The rapid exchange within the undocked states (UD↔UP) repeatedly juxtaposes the two loops, increasing the probability of interaction between them and thus folding. Loop–loop interaction is clearly a complex process, involving many specific contacts , and the local conformation of the loops in the folded ribozyme is significantly altered from that in the isolated loops [9,10]. It is therefore likely to require multiple conformational adjustments to achieve the active state. The UD↔UP transition occurs at a rate of approx. 100 s−1 in 0.5 mM Mg2+, whereas the rate of formation of the F state is significantly lower at ≤3 s−1. The folding under these conditions must be limited by the conformational changes involved in loop–loop interaction rather than by the junction dynamics.
The rate of loop docking is approx. 500-fold higher than that of the equivalent process measured for the minimal-hinged form of the ribozyme that lacks the four-way junction . Thus the junction accelerates the rate of folding of the ribozyme into the active conformation, so that folding is no longer rate-limiting as it is for the minimal form. Secondly, the junction ensures that folding occurs under physiological ionic conditions , again in contrast with the minimal form. The helical junction is not essential for activity, since the minimal ribozyme exhibits good cleavage activity, but it is important for efficient activity under cellular conditions. This situation is similar to that described for the hammerhead ribozyme, where loops in two helical stems promote activity at physiological ionic concentrations  and greatly facilitate folding . These auxiliary elements, the loops of the hammerhead and the junction of the hairpin ribozyme, appear to act as ‘folding enhancers’.
The cleavage and ligation reactions of the hairpin ribozyme
Single-molecule FRET studies provide the means to observe rates of ribozyme reactions, which are free of uncertainties about the processes of undocking and product release. This is made possible by the very different dynamics of the intact ribozyme and its product of cleavage. Figure 4 shows an experimental setup, where we immobilize a ribozyme in which a shortened 3′-end of the d strand limits the extent of the terminal helix of arm A to 3 bp. Thus, on cleaving, the 3′-product dissociation is rapid, and the remaining species is expected to behave like a simple junction. A typical time trace is shown. On addition of 10 mM Mg2+, the ribozyme folds into a stable high-FRET state for a period of time, after which it spontaneously changes behaviour with the onset of rapid two-state fluctuations. The latter state is typical of a simple junction, and we therefore conclude that a cleavage reaction has occurred to generate that species. The inverse of the time between docking and the emergence of junction behaviour is a measure of the apparent cleavage rate. Analysis of a series of reactions gave a cleavage rate of approx. 1 min−1 under these conditions.
This experiment still leaves some uncertainty in terms of the rate of undocking following the cleavage reaction. To determine the rates of undocking and ligation, we studied a ribozyme with a longer substrate strand, such that the terminal helix of arm A is increased to 7 bp, so that dissociation of the 3′-cleavage product should be slow (Figure 5). On addition of Mg2+ ions, these ribozymes exhibit switching between two distinct dynamic modes. At some times, the molecules remain stably docked, whereas at other times they display rapid docking and undocking transitions. We assign the two dynamic modes (on the basis of these and other experiments) to the ligated and cleaved forms of the ribozyme respectively. The docking and undocking rates within the cleaved state are kCdock=2.5(±0.1) s−1 and kC, obsundock=2.3(±0.1) s−1. These rates are an order of magnitude slower than the junction dynamics under the same conditions.
These experiments allow us to calculate the internal cleavage and ligation rates for the hairpin ribozyme. The internal cleavage rate (kC) is close to 0.6 min−1 in the presence of 1 mM Mg2+. The internal ligation rate (kL, defined as the rate of ligation in the docked ribozyme) is approx. 0.3 s−1. The rates of reaction and docking/undocking are summarized in Figure 6. The reaction is significantly biased towards ligation, with an internal equilibrium constant of Kint=kL/kC=34. This is important to maintain the integrity of the circular (−) strand, while it serves as a template for (+) strand synthesis. However, cleavage of the concatenated product of the first round of replication is possible because of the rapid undocking that follows cleavage.
How does the hairpin ribozyme catalyse phosphoryl transfer reactions?
Inspection of the crystal structure (Figure 7) of the hairpin ribozyme with a non-cleavable substrate  and a transition state inhibitor  suggests some strong candidates for catalytic groups. G+1 located adjacent to the cleavage site is pulled out of the A loop by interaction with C25 in a pocket in the B loop. The net effect is to alter the conformation of the backbone leading to a position of the 2′-hydroxy group so that it is well aligned for in-line nucleophilic attack on the 2′-phosphorus. This is catalytically useful, but probably cannot provide more than a 100-fold increase in reaction rate by itself. Two nucleobases, G8 from the A loop and A38 from the B loop, are closely juxtaposed at the centre of activity and hydrogen-bonded to phosphate oxygen atoms . These nucleobases could act in general acid–base chemistry. G8 is positioned to remove the proton from the 2′-hydroxy group, which would greatly improve its nucleophilicity in the cleavage reaction, whereas A38 is in position to donate a proton to the 5′-oxyanion leaving group. Of course, by the principle of microscopic reversibility, if G8 and A38 act as general base and acid respectively in the cleavage reaction, they will be acid and base respectively in the ligation reaction also. If we assume pKa values of 6 (a perturbed but realistic value) and 9.5 for A38 and G8 respectively, this should lead to rates of both cleavage and ligation that increase at low pH and then plateau at approx. pH 7 (this kind of analysis has been well described by Bevilacqua ). The shape of both curves will reflect the pKa of A38 according to this scheme. We have measured both the rates as a function of pH. The rate of ligation was measured using the single-molecule experiments and the rate of cleavage by bulk methods under conditions where the single-molecule experiments have taught us that the folding is not rate-limiting. Both reactions exhibit very similar pH dependence (Figure 8), corresponding to the titration of a group with a pKa 6.2 (ligation) and 6.3 (cleavage). This is certainly within the range of an adenine residue with a perturbed pKa, but cannot prove the point by itself.
Comparison with the VS (Varkud satellite) ribozyme
It is useful to compare the hairpin ribozyme with another nucleolytic ribozyme studied in this laboratory, namely the VS ribozyme. The VS ribozyme is the largest of this class and the only one for which there is no crystal structure available at present. However, we have determined the global fold [15,16], the probable manner of the interaction with the substrate , the location of the active site [17⇓–19] and a strong candidate for a participating nucleobase [20,21]. The global conformation comprises two three-way helical junctions. This generates a cleft between two helices, which appears to be the primary binding site for the substrate stem-loop, whereupon it can interact with the active site, the A730 loop [16,22]. Within the loop, A756 appears to play a key role , and there is evidence that the reactivity of the ribozyme is sensitive to its state of protonation . The ligation rate varies with pH in a manner consistent with the titration of a group with a pKa value of 5.6 . Thus in both, the hairpin and VS ribozymes, the environment in which catalysis can occur seems to be generated by an intimate loop–loop association, and it seems probable that both ribozymes exploit general acid–base catalysis mediated by nucleobases.
We thank Cancer Research U.K. and National Science Foundation for support of our research in Dundee and Urbana respectively.
RNA Structure and Function: Joint Biochemical Society/Royal Society of Chemistry Focused Meeting held at the Michael Swann Building, University of Edinburgh, U.K., 4–6 December 2004. Organized and Edited by S.V. Graham (Glasgow, U.K.) and D.M.J. Lilley (Dundee, U.K.). Sponsored by BBSRC (Biotechnology and Biological Sciences Research Council), Glen Research, Promega UK Ltd, VH Bio Ltd, Stratagene, New England Biolabs (UK) Ltd, MWG Biotech UK Ltd, Ambion Europe Ltd and Link Technologies Ltd.
Abbreviations: FRET, fluorescence resonance energy transfer; VS, Varkud satellite
- © 2005 The Biochemical Society