Solution structure of the loop B domain from the hairpin ribozyme

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson-Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme-substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.     

Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.

The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.     

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.     

Investigation of the proposed interdomain ribose zipper in hairpin ribozyme cleavage using 2'-modified nucleosides

The hairpin ribozyme achieves catalytic cleavage through interaction of essential nucleotides located in two distinct helical domains that include internal loops. Initial docking of the two domains is ion dependent and appears to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The proposed structural rearrangement may also be ion dependent and is now of increased importance due to recent evidence that docking is not rate limiting and that metal ions are unlikely to be involved in the chemical cleavage step. An initial structural model of the docked hairpin ribozyme included a proposal for a ribose zipper motif that involves two pairs of hydroxyl groups at A(10) and G(11) in domain A pairing with C(25) and A(24) in domain B, respectively. We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the active, conformationally rearranged ribozyme that is fit for cleavage. We have chemically synthesized a series of individually modified hairpin ribozymes containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deoxy-2'-fluoro at each of the four nucleoside positions, 2'-amino-2'-deoxy, 2'-deoxy-2'-thio, and 2'-arabino at position C(25), and 2'-oxyamino at position A(10), as well as some double substitutions, and we studied their cleavage rates under both single- and multiple-turnover conditions. We conclude that at least some of the hydrogen-bonding interactions in the ribose zipper motif, either as originally proposed or in a recently suggested structural variation, are unlikely to be present in the active rearranged form of the ribozyme that undergoes cleavage. Instead, we provide strong evidence for a very precise conformational positioning for the residue C(25) in the active hairpin. A precise conformational requirement would be expected for C(25) if it rearranges to form a base-triple with A(9) and the essential residue neighboring the cleavage site G(+1), as recently proposed by another laboratory. Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.     

A conformational change in the "loop E-like" motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis

The catalysis of site-specific RNA cleavage and ligation by the hairpin ribozyme requires the formation of a tertiary interaction between two independently folded internal loop domains, A and B. Within the B domain, a tertiary structure has been identified, known as the loop E motif, that has been observed in many naturally occurring RNAs. One characteristic of this motif is a partial cross-strand stack of a G residue on a U residue. In a few cases, including loop B of the hairpin ribozyme, this unusual arrangement gives rise to photoreactivity. In the hairpin, G21 and U42 can be UV cross-linked. Here we show that docking of the two domains correlates very strongly with a loss of UV reactivity of these bases. The rate of the loss of photoreactivity during folding is in close agreement with the kinetics of interdomain docking as determined by hydroxyl-radical footprinting and fluorescence resonance energy transfer (FRET). Fixing the structure of the complex in the cross-linked form results in an inability of the two domains to dock and catalyze the cleavage reaction, suggesting that the conformational change is essential for catalysis.     

Use of both direct and indirect 13C tags for probing nitrogen interactions in hairpin ribozyme models by 15N NMR

We have used the synthesis and 15N NMR study of separate loop A and loop B domains of the hairpin ribozyme to demonstrate that multiple 15N atoms can be incorporated into an RNA strand and be unambiguously distinguished through a combination of direct and indirect tagging by 13C atoms. Absence of 15N chemical shift changes shows that the G8N1 in loop A does not become deprotonated up to pH 8, and that the G21N7 of loop B does not bind to Mg2+.     

The loop B domain is physically separable from the loop A domain in the hairpin ribozyme.

In order to understand the catalysis mechanism of the hairpin ribozyme, mutant ribozymes were constructed. The distance between the loop A domain and the loop B domain was extended by inserting various lengths of nucleotide linkers at the hinge region in cis mutants, or the domains were separated physically in a trans mutant. All the mutant ribozymes, including the trans mutant, could cleave substrate RNA at the predicted site. A cis mutant with a single nucleotide insertion exhibited cleavage activity about twice as high as that of the wild-type (wt) ribozyme. The insertion of 2-5 nucleotides (nt) gradually reduced the activity to the level of the wt ribozyme. Insertion of a longer linker, up to 11 nt, resulted in the reduction of activity to one half of that of the wt ribozyme. The ribozyme with a single nucleotide insertion at the hinge region seems to form a more suitable conformation for catalysis by three-dimensional fold-back of the loop B to loop A containing the cleavage site. The trans mutant, in which the A and B domains were physically separated, maintained a significant level of activity, suggesting that both domains are necessary for catalysis, but separable. These results demonstrate that interaction between the A and B domains results in catalysis.     

Effects of base substitutions in an RNA hairpin from molecular dynamics and free energy simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.     

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.     

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.     

Dynamics and stability of GCAA tetraloops with 2-aminopurine and purine substitutions

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G-->AP) and the third (A-->PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A-->PUR) or in the C-G base pair closing the tetraloop (G-->AP) to some extent influenced the loop structure and dynamics. These loops did not display the long-lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the glycosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

In vitro selection of second site revertants analysis of the hairpin ribozyme active site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.     

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

Abstract

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

Determination of metal ion binding sites within the hairpin ribozyme domains by NMR

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.     

In vitro selection of hairpin ribozymes activated with short oligonucleotides

We have carried out an in vitro selection to obtain an allosteric hairpin ribozyme, which has cleavage activity in the presence of an exogenous short oligonucleotide as a regulator. Random sequences were inserted in a region corresponding to the hairpin loop of the ribozyme. After 12 rounds of selection, DNA templates were cloned. Of a total of 34 clones, 18 contained the same sequence, and the obtained hairpin ribozymes showed the cleavage activity specifically in the presence of the regulator oligonucleotide. All of the clones contained sequences complementary to the regulator oligonucleotide. The ribozymes with high cleavage activities gained characteristic hairpin loops at the random domain, which were similar to each other. In the absence of the oligonucleotide, the loop domain within the allosteric ribozyme probably forms a slipped hairpin loop, and the complementary sequence, with the regulator oligonucleotide located at the single stranded loop, would allow easy access of the oligonucleotide. The binding of the regulator oligonucleotide triggers a structural change of the hairpin loop to form an active conformation. Furthermore, we constructed an allosteric hammerhead ribozyme by introducing the characteristic hairpin loop. The modified hammerhead ribozyme was also changed to an allosteric ribozyme, which was activated by the addition of the regulator oligonucleotide. The characteristic hairpin loop, which was proved to be regulated by an exogenous oligonucleotide in this report, may be used to control RNA functions in various fields.     

Adenine protonation in domain B of the hairpin ribozyme

Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.     

Structural basis for the guanosine requirement of the hairpin ribozyme

To form a catalytically active complex, the essential nucleotides of the hairpin ribozyme, embedded within the internal loops of the two domains, must interact with one another. Little is known about the nature of these essential interdomain interactions. In the work presented here, we have used recent topographical constraints and other biochemical data in conjunction with molecular modeling (constraint-satisfaction program MC-SYM) to generate testable models of interdomain interactions. Visual analysis of the generated models has revealed a potential interdomain base pair between the conserved guanosine immediately downstream of the reactive phosphodiester (G(+1)) and C(25) within the large domain. We have tested this former model through activity assays, using all 16 combinations of bases at positions +1 and 25. When the standard ribozyme was used, catalytic activity was severely suppressed with substrates containing U(+1), C(+1), or A(+1). Similarly, mutations of the putative pairing partner (C(25) to A(25) or G(25)) reduce activity by several orders of magnitude. The U(25) substitution retains a significant level of activity, consistent with the possible formation of a G.U wobble pair. Strikingly, when combinations of Watson-Crick (or wobble) base pairs were introduced in these positions, catalytic activity was restored, strongly suggesting the existence of the proposed interaction. These results provide a structural basis for the guanosine requirement of this ribozyme and indicate that the hairpin ribozyme can now be engineered to cleave a wider range of RNA sequences.     

Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance.

The hairpin ribozyme derived from the minus strand of the satellite RNA associated with the tobacco ringspot virus is one of the small catalytic RNAs that has been shown to catalyze trans-cleavage reactions. There is much interest in designing hairpin ribozymes with improved catalytic activity for the development of new therapeutic agents. Extensive mutagenesis studies as well as in vitro selection experiments have been performed to define the structure and optimize its catalytic activity. This communication describes a comparative kinetic analysis of four structural variants, introduced, either alone, or in combination, into the hairpin ribozyme. We have shown that extension of the helix 2 from 4 to 6 bp resulted in a significant decrease in K(M). Furthermore, the combination of this extension with the simultaneous stabilization of helix 4, led to a more than two-fold increase in the catalytic efficiency. This variant showed a 15-fold reduction in the K(M) value in respect to the wild-type ribozyme. This could be of great interest for the in vivo application of this catalytic motif. The 9-bp enlargement of helix 4 implied about a three-fold improvement in the catalytic activity. Similarly, the U39C substitution brought up the efficiency of the ribozyme slightly. However, introduction of nucleotides at the hinge region between A and B domains reduced the catalytic activity. This reduction was gradually increased with the number of nucleotides. Results obtained with variants carrying more than one modification always agreed with the ones obtained from each single variant.