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J. Biol. Chem., Vol. 275, Issue 41, 32157-32166, October 13, 2000

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Analysis of the Functional Role of a G·A Sheared Base Pair by in Vitro Genetics^*

Bruno Sargueil^§, Jeffrey McKenna^¶, and John M. Burke^¶

From the  Centre de Génétique Moléculaire, CNRS, Avenue de la Terrasse, 91190 Gif sur Yvette, France and the ^¶ Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405

Received for publication, June 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A classical genetic strategy has been combined with an in vitro selection method to search for functional interactions between the two domains of the hairpin ribozyme. G²¹ is located within internal loop B; it is proposed to form a sheared base pair with A⁴³ across loop B and to bind a Mg²⁺ ion. Both nucleotides are important for ribozyme function, and G·A sheared base pairs are a very widespread motif in structured RNA. We took advantage of its presence in the hairpin ribozyme to study its functional role. Pseudorevertants, in which the loss of G²¹ was compensated by mutations at other positions, were isolated by in vitro selection. The vast majority of G²¹ revertants contained substitutions within domain A, pointing to functional communication between specific sites within the two domains of the hairpin ribozyme. The possibility of a direct or redundant contacts is supported by electrophoretic mobility shift studies showing that a complex formed between domain B of the ribozyme and the substrate was disrupted and restored by base substitutions that have analogous effects on catalytic activity. The functional significance of this complex, the role of the nucleotides involved, and the basis for magnesium ion requirement is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Four natural small catalytic RNA motifs are known to mediate cleavage and ligation, reactions central to RNA processing events associated with replication of the genomes of certain RNA viruses or virus-like RNA replicons. These include the hairpin (1-3) hammerhead (4, 5), hepatitis  (6), and Varkud satellite (7) ribozymes (also see Ref. 8). Divalent cations including Mg²⁺ are known to support catalysis. However, recent evidence indicates that divalent metal ions function to facilitate ribozyme folding but do not play a direct obligatory role in catalysis for the hairpin, hammerhead, and VS ribozymes (9-12). For each of these three ribozymes, it appears that catalytic function is resident in the folded structure of the RNA itself.

The 21-kDa complex between the 50-nucleotide hairpin ribozyme and its 14-nucleotide substrate consists of four short helical elements and two internal loops (Fig. 1). The complex is organized into two domains. Domain A consists of the substrate and substrate-binding sequence, forming helix 1, internal loop A, and helix 2. Domain B is located entirely within the ribozyme and consists of helix 3, internal loop B, and helix 4. The two domains of the ribozyme-substrate complex interact through tertiary contacts to generate a complex necessary for catalytic activity. This was initially established through linker insertion studies (13, 14) and domain separation and reconstitution experiments (15). Covalent cross-linking studies were used to develop a preliminary tertiary structure model for the ribozyme-substrate complex (16). Subsequently, fluorescence resonance energy transfer analysis (17, 18), and hydroxyl radical protection experiments (19) were used to establish the requirements for docking of the two domains and to map the interface between the domains in the docked complex.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   In vitro selection of pseudorevertants of G21 mutants. Secondary structure of the ribozyme-substrate complex is shown; the indications of noncanonical pairings are from Ref. 50 for loop A and Ref. 22 for loop B. Boldface type indicates sites at which sequence variation was introduced into the initial population of RNA variants, as described under "Materials and Methods". Nucleotides in the ribozyme are numbered 1-50. Substrate nucleotides to the 5' side of the cleavage-ligation site (arrow) are indicated with negative numbers; those to the 3' side have positive numbers. RT-PCR, reverse transcription-polymerase chain reaction.

G²¹ is located at a particularly interesting location within the hairpin ribozyme. Ultraviolet irradiation at low intensities induces a specific photo-cross-link between G²¹ and U⁴² (20). Although the cross-linked RNA has little or no catalytic activity (20), the cross-linkable conformation is likely to represent a ground state structure of the active ribozyme species, because 1) 90% cross-linking can be achieved with molecules that fold homogeneously and 2) domain B modifications that increase cross-linking yield concomitantly increased catalytic activity (21).¹ The region of loop B in which G²¹ is embedded represents a common folding motif, also found within loop E of eukaryotic 5 S rRNA, the conserved central domain of viroids, and the sarcin-ricin loop of large rRNA (20, 22-24). Within this motif, a local reversal of strand direction results in cross-strand stacking of the two cross-linkable bases. In the context of the hairpin ribozyme, G²¹ forms a sheared base pair with A⁴³ and stacks on U⁴² (Fig. 2). A remarkable feature of this structure is that both the major and minor grooves are wide, and the Watson-Crick base pairing faces of some of the bases are left unpaired and are projected into the grooves. This structural model has recently been confirmed by the NMR-derived structure of the isolated loop B (22). It has been proposed that this motif is well suited for specific binding of protein or RNA species (25). Particularly noteworthy is the observation that N-7, N-1, and N-2 of G²¹ are protected from chemical modification upon Mg²⁺ addition (22). This could be related to the recent finding that a Mn²⁺ ion can bind in between A²⁰ and G²¹ (26).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   G21·A43 sheared base pairs within internal loop B. This structural model has been developed from NMR spectroscopy data (22, 26). Black, G21; green, A43; gold, A20; gray, C44. A, stereoview of the G21·A43 and A20·C44 base pairs. The hexahydrated Mg2+ ion is shown bound as modeled in Ref. 26. B, stereoview of loop B global architecture. A20·C44 and G21·A43 pairings and the related metal ion binding site are shown. This figure has been elaborated using the Ribbons software and the coordinates from Ref. 26.

The identification of specific tertiary contacts between the two domains presents a very challenging problem. Several biochemical and biophysical strategies have been used successfully to probe the tertiary structure of small ribozymes. Recently, an interaction between the nucleotide G⁺¹ of the substrate and C²⁵ has been biochemically identified (27).

Here, we describe a fundamentally different strategy, based on a classical genetic approach, to search for functional interactions between the two domains of the hairpin ribozyme. The method is in vitro selection of pseudorevertants of loss of function mutations. In genetically tractable biological systems, the selection and analysis of second site revertants has been widely applied to examine functional interactions between molecules and within different parts of the same molecules. Advantages of the in vitro adaptation of this strategy to RNA structure are, first, that the investigator has complete control over the location, distribution, and frequency of mutations and, second, that the selection can be performed with very many more variants than can be accommodated in an in vivo selection. Our results clearly demonstrate functional compensation between specific sites within the two domains of the hairpin ribozyme. A detailed study allowed us to assess the function of the G·A sheared base pair and to understand the role of one of the magnesium ions required for activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

RNA Preparation-- RNA was transcribed with T7 RNA polymerase using synthetic oligonucleotide templates (28). Partially double-stranded oligonucleotides (200 pmol) were transcribed by T7 RNA polymerase at 37 °C for 3 h in 1-ml reactions containing 40 mM Tris-HCl (pH 8), 25 mM MgCl₂, 2 mM NTPs, 1 mM spermidine, and 5 mM dithiothreitol. Transcripts were purified by electrophoresis through 20% polyacrylamide, 7 M urea gels, eluted by diffusion, precipitated, and quantitated by UV absorbance. Body-labeled substrate RNA was prepared as described above, except that 100 pmol of template was used in a 100-µl reaction containing 50 pmol of [-³²P]CTP and 0.5 mM unlabeled CTP. Following gel purification, substrates were quantified by measuring radioisotope incorporation.

Mixed RNA-DNA oligonucleotides were synthesized by solid-phase synthesis as described (29). End-labeling of RNA molecules was accomplished by dephosphorylation of 5'-ends using calf intestinal alkaline phosphatase (1 unit/50 pmol of RNA) for 1 h at 50 °C, followed by phosphorylation with T4 polynucleotide kinase (2 units/10 pmol of RNA) using 25 pmol of [-³²P]ATP and purified as described above.

Activity Assays-- Cleavage assays were conducted using hairpin ribozymes synthesized in two segments, as described (30). The 5' and 3' ribozyme segments were mixed at a concentration of 100 nM, denatured for 1 min at 90 °C, and then renatured for 20 min on ice in cleavage buffer (5 or 12 mM MgCl₂ (as indicated), 40 mM Tris-HCl (pH 7.5)). To assay a large number of mutants under standardized conditions, we examined cleavage time courses under the following multiple turnover conditions, which allows us to have a reasonable estimate of the cleavage efficiency over a wide range of activity. In these experiments, 20 nM ribozyme and 200 nM substrate were incubated at 37 °C in cleavage buffer (5 or 12 mM MgCl₂ (as indicated), 40 mM Tris-HCl (pH 7.5)), reactions were initiated by mixing an equal volume of ribozyme and substrate, and aliquots were removed and quenched at 0, 5, 10, 15, 30, and 60 min. Reaction products were separated using a 20% denaturing polyacrylamide gel, and dried gels were quantified using a Bio-Rad GS-250 Molecular Imager. To determine initial reaction rates, results were fitted by linear regression to the equation ln F = kt + b, where F is the unreacted fraction and t is the reaction time. Each mutant was assayed side by side with the wild type construct and relative initial rate constants (k_rel = k_mutant/k_wt) were calculated. Each construct was assayed at least twice. Absolute initial rates varied by up to 30% from experiment to experiment, but the reported relative values were reproducible to within a 10% error, usually less. Mg²⁺ dependence was determined using the cleavage protocol described above but using single turnover conditions (200 nM ribozyme, 20 nM substrate); the Mg²⁺ concentrations used were 2, 5, 10, 20, 50, and 100 mM, and depending on the efficiency of the considered ribozyme, aliquots were taken at 0, 2, 5, 10, and 15 min or at 0, 5, 10, 15, 30, and 60 min.

Photo-cross-linking-- RNA cross-linking was performed as described in Ref. 20. Gel-purified radiolabeled RNA was denatured at 70 °C for 2 min and then renatured for 20 min on ice in cleavage buffer (12 mM MgCl₂). Twenty-microliter aliquots were irradiated at a distance of 1 cm with a 254-nm hand-held ultraviolet light source. Irradiated samples were then analyzed by electrophoresis through 15% denaturing polyacrylamide-urea gels, and reaction products were quantitated as described above. Cross-linked nucleotides were mapped using end-labeled RNA and partial alkaline hydrolysis and identified by electrophoresis alongside enzymatic RNA sequencing ladders (20).

In Vitro Selection-- The general method was carried out as described (31, 32). The transcriptional template was generated by annealing and extension of two overlapping oligonucleotides, a 74-nucleotide segment containing the coding sequences for a T7 promoter, a primer binding site, part of the ribozyme sequences (5'-TAATACGACTCACTATAGGACGCACGTGAGCTCGAGTAAACAGAGAANNCACCCAGA(ATC)AAACACACGTTGTGGT-3'), and a 63-nucleotide segment representing the antisense sequences for the remainder of the ribozyme, a linker, the substrate, and a second primer binding site (5'-CGACGTCGGCTCTAGAGAAACAGGACTNNCAGAGATCTGTACCAGGTAATATACCACAACGTG-3'). Overlapping sequences are in boldface type. Underlined nucleotides were mutagenized to a total frequency of 12% (i.e. each mutant base was present at a frequency of 4%). The double-stranded template (400 pmol) was transcribed with T7 RNA polymerase for 3 h; these conditions are permissive for self-cleavage of the wild type molecule and active variants. Cleaved (active) and uncleaved (inactive) molecules were separated on a 8% polyacrylamide, 7 M urea gel and eluted. For the active pool, ligation reactions were carried out by incubating the active fraction with 200 pmol of a substrate for ribozyme ligation in cleavage buffer (12 mM MgCl₂) at 0 °C for 30 min. The oligonucleotide substrate for ligation was a DNA-RNA hybrid containing a binding site for primer 2 (P2, 5'-GUCCUGUUUTCTAGAGACGCCTGCTG-3'; ribonucleotides in boldface type). The ligation product was isolated using a 8% polyacrylamide, 7 M urea gel, reverse-transcribed, and amplified by polymerase chain reaction using the primers P1 (5'-CACGAGGCGTCTCTAGA-3') and P2 (5'-TAATACGACTCACTAGGACGCACGTGAGCTCG-3'). Inactive molecules were reverse transcribed and amplified using primers P2 and P3 (5'-CGACGTCGGCTCTAGAG-3').

Gel Shift Assay-- Substrate and loop B domain interactions were analyzed using a gel shift assay essentially as described previously (33). Trace amounts of 5'-radiolabeled substrate (<1 nM) were incubated in siliconized tubes with increasing amounts of unlabeled loop B domain (0, 0.1, 0.5, 1, 2, 5, 10, and 20 µM). The mix was incubated for 20 min at 37 °C, and binding was allowed to proceed for 2 h on ice. The 37 °C 20-min incubation allows the loop B domain to reach the same folding state as a "standard" denaturation renaturation (90 °C for 1 min followed by 20 min on ice), but avoids dimer formation as monitored with a cross-link assay (20).² After the addition of 5% glycerol, samples were run on a 15% nondenaturing polyacrylamide gel containing 25 mM magnesium acetate and 40 mM Tris acetate, pH 7, for 16 h at 11 watts and 4 °C. The results were quantitated using a Bio-Rad GS 250 molecular imager. The proportion of bound substrate as a function of concentration of loop B domain was plotted on a semilogarithmic scale, and the K_D value was estimated as the observed concentration corresponding to half of the binding at saturation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

G²¹ Mutants Strongly Inhibit Cleavage-- In vitro selection experiments showed that G at ribozyme position 21 is important for cleavage (34). However, selection experiments generally are of limited utility in understanding defects associated with specific mutations. Typically, only a small fraction of inactive clones are recovered, and inactive variants usually contain multiple base substitutions. To begin analysis of the functional importance of G²¹, we synthesized ribozymes containing each of the four possible bases at position 21, and conducted parallel trans-cleavage assays. G²¹ substitutions reduced cleavage efficiency by 10-100-fold in a cleavage buffer containing 5 mM Mg²⁺, with U being the most highly inhibitory (Table I; see also Refs. 35 and 36). Partial suppression of the cleavage deficiencies was observed at 12 mM Mg²⁺ for all three mutants. To better evaluate the effect of ions, we carried out a study to compare the magnesium dependence of cleavage for the 21GU mutant and the WT.³ The apparent K_Mg²⁺ for the WT ribozyme is 15 mM, and the saturation is obtained by 50 mM. These values are in good agreement with the values found in other studies using the same sequences (17, 37, 38). The requirement is clearly increased for the 21GU mutant for which the K_Mg²⁺ is about 30 mM, although a level of activity comparable with the WT molecule is reached over 100 mM.

When analyzed on a non denaturing polyacrylamide gel, the WT ribozyme-substrate complex run as two bands. The fastest migrating band corresponds to the active "docked" ribozyme, while the low mobility complex was shown to be an inactive complex in which domain A is stacked over domain B (39, 40). When conducted with the 21GU ribozyme, the gel shift mobility assay in the presence of 5 mM Mg²⁺ showed that most of the complex is undocked (data not shown).

Mutations at A⁴³, the pairing partner of G²¹ in the loop B structure, indicated that a purine is required at this position. 43AG shows only a 2-3-fold reduction in cleavage activity. However, no cleavage was detectable during analysis of the 43AC and 43AU mutation (see also Refs. 31 and 35-37).

It is striking that the most inhibitory substitutions at positions 21 and 43 are those with the potential to generate canonical base pairs (21GU, 43AC, and 43AU). These results could reflect that the formation of a 21·43 Watson-Crick base pair induces an inactive conformation, possibly by collapsing the helix 3 proximal segment of internal loop B into an A-form extension of helix 3.

In Vitro Selection of Second Site Revertants of G²¹ Mutations-- In vitro selection was used to search for second site revertants that could compensate for mutation of G²¹ to A, U, or C (Fig. 1). The synthetic DNA template encoded a complex population of sequence variants containing equimolar quantities of A, U, and C at position 21, while G was absent. In addition, we mutagenized the 19 functionally important bases within internal loops A and B to a frequency of two additional mutations per molecule and completely randomized the two base pairs of helix 2 that are proximal to internal loop A (see "Materials and Methods"). The mutation 39UC had previously been isolated as a nonspecific suppressor of several ribozyme mutations (31, 34, 41). To avoid the potential recovery of this previously characterized suppressor, we did not mutagenize U³⁹ into the population before initiating selection. This design yields a pool of variant molecules with a complexity of approximately 10⁶ unique sequences (42). To prevent recovery of a true revertant (G²¹) due to polymerase misincorporation, a single round of selection was performed. Previous experience shows that a single round selection for active variants provides a very high level of enrichment for DNA encoding active ribozymes and that the selected population consists of both highly active and a variety of suboptimal species.⁴ It should be noted that a round of selection provides two selective steps for RNA catalysis, cleavage followed by ligation.

The DNA template pool was transcribed for 3 h under conditions favorable for self-cleavage; then cleaved (active) and uncleaved (inactive) species were separated by preparative gel electrophoresis. To empirically assess the sites and frequencies of sequence variation in the pool, cDNA from inactive transcripts was synthesized, cloned, and sequenced. The frequency and position of mutations were consistent with those predicted by design of the variant pool. To recover the active populations, molecules that had undergone cleavage were allowed to carry out RNA-catalyzed ligation, and then cDNA copies of active ribozymes were synthesized, amplified, cloned, and sequenced.

Cloned cDNA molecules from the active pool were characterized by sequencing and by monitoring the amount of self-cleavage during in vitro transcription (reaction including 20 mM MgCl₂ and 2 mM spermidine). Representative clones derived from the active pool are shown in Table II. As expected, all recovered clones contained G²¹ substitutions. A small number of clones containing only G²¹ mutations were identified and showed the very low levels of self-cleavage activity expected from the trans-cleavage assays described above. A number of pseudorevertants were identified that displayed self-cleavage activities much higher than those with G²¹ mutations alone. Most of these clones had dual base substitutions at the 3·12 base pair within helix 2, previously shown to be essential for substrate binding and cleavage (43). In these clones, G²¹ mutations to U, C, and A were accompanied by changes of the a³·U¹² base pair to g·C, c·G, and u·G. In addition, a single pseudorevertant was identified, which contained a transversion of the base immediately 5' of G²¹, 20AC.

Second Site Mutations in Helix 2 Base Pair (3·12) Rescue G²¹ Mutations-- The functional compensation of G²¹ mutants by substitutions at the 3·12 base pair is particularly interesting because the compensatory mutations lie in the loop A domain of the ribozyme, while the original mutation is in the loop B domain. Trans-cleavage assays using synthetic ribozymes and oligonucleotide substrates were carried out in order to examine more closely the efficiency and specificity of this compensatory effect. Using the two-piece ribozyme construct previously described and characterized (30, 37), we generated ribozymes and substrates to test all possible combinations of bases at position 21 and base pairs at 3·12. Results are shown in Fig. 3 and Table III (please note that helix 4 sequence is 5'-C₂₇ACC-3' and 5'-GGUG₃₅-3' except for the simple G²¹ mutants that have a 2-G·C-base pair extended helix 4, which slightly enhances their activity (21) and therefore slightly undermines their mutant phenotype).

View larger version (59K): [in this window] [in a new window]   Fig. 3.   Base pair substitutions in helix 2 suppress G21 mutations. Autoradiogram of trans-cleavage time courses, conducted in a buffer containing 5 mM MgCl2, as described under "Materials and Methods" and in the legend to Table I.

View this table: [in this window] [in a new window]   Table III 3a  g/12U  C supresses G21 mutations Trans-cleavage time courses were conducted in a buffer containing 5 mM MgCl2, and cleavage rates were determined and normalized to the wild type rate as described under "Materials and Methods."

Because it is the most strongly inhibited, we will first discuss the 21GU mutant. The autoradiograms of Fig. 3 indicate that changing the a³·U¹² base pair to g·C dramatically increases the activity of the 21GU mutant. The increase in the initial cleavage rate is over 100-fold in the presence of 5 mM MgCl₂ (Table III). This same base pair substitution also increases the initial cleavage rate of the G²¹ ribozyme, but by a factor of 3 only. Interestingly, all base pair substitutions at 3·12 enhance the activity of the 21GU mutant ribozyme. Rate enhancement varies from 7- to 110-fold. A similar pattern is observed when G²¹ is changed to A or to C. For each G²¹ mutation, the naturally occurring base pair (a³·U¹²) is the least favorable for cleavage. For 21GA and 21GC, base pair substitutions at 3·12 increase initial cleavage rates by factors of 2-16 relative to a³·U¹². In every case, g³·C¹² has the most significant effect.

Regardless of the identity of the base at position 21, cleavage is strongly inhibited by mutational disruption of the 3·12 base pair (data not shown). This result is consistent with the selection experiment described above, in which all active variants recovered could form 3·12 base pairs, and is also fully consistent with previous selection and mutational studies (43, 44).

To screen for potential backbone interactions involving nucleotides G²¹, a³, and U¹², the effects of substitutions with 2'-deoxyribonucleotides at these three sites were examined. None had a strong effect; deoxyribose substitution at positions G²¹, a³, and U¹² reduced initial cleavage rates by 2-fold, no reduction, and 4-fold, respectively (data not shown, and see Refs. 45 and 46). We conclude that only U¹² 2'-OH could potentially be involved in the rescue phenomenon observed.

Another suppressor of G²¹ mutations has previously been isolated (31, 34, 41). To analyze the possibility of an interaction between U³⁹ and the 3·12 base pair, we combined mutations at these positions together with the 21GU mutation. 39UC appeared to have a very marginal effect compared with g³·C¹². We concluded that these two suppressors act in different ways.

Finally, the magnesium requirement for cleavage was determined for the 12UC and 21GU/12UC ribozymes (Fig. 4). The K_Mg²⁺(app) for the 12UC construct is 3-4 mM, and the saturation is obtained at about 10 mM. Once more, this is in good agreement with the data obtained previously by others using the 12UC variant of the WT ribozyme (47, 48). The revertant shows a similar behavior except that the K_Mg²⁺(app) value is slightly higher (about 6 mM).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Influence of G21 and a3·U12 mutations on the magnesium dependence of cleavage rate of G21 and a3·U12 mutant ribozymes. Single turnover reactions were carried out as described under "Materials and Methods" in the presence of increasing amounts of MgCl2. The values reported are the cleavage rate constant relative to the wild type construct.

Partial Suppression of 21GU by 20AC-- Independent of the 3·12 substitutions, the mutation 20AC was recovered as a second site revertant of the 21GU mutation and showed a significant increase in self-cleavage activity relative to 21GU. Cleavage assays using trans-acting ribozymes were conducted and confirmed that 20AC is indeed a second site suppressor of 21GU, increasing activity by a factor of approximately 15 (Table IV, top).

View this table: [in this window] [in a new window]   Table IV 20A  C is a suppressor of 21G  U The effect of A20 and G21 substitutions in the context of a wild type base pair (a3 · U12) in helix 2 is shown at the top. Cleavage rates were determined and normalized to the wild type rate, as described under "Materials and Methods" and in the legend to Table I. The effect of A20 substitutions and 3 · 12 base pair substitutions in the context of a 21G  U mutant background is shown at the bottom. ND, not determined.

To explore the specificity of this suppression, we synthesized and analyzed a panel of ribozymes with mutations within loop B at positions 20 and 21. In the context of the wild type ribozyme, all mutations of A²⁰ are slightly inhibitory (Table IV, top). In the 21GU background, the only significant compensatory effect observed was that of the original isolate, 21G U/20AC.

To investigate the possibility of a direct contact between A²⁰ and the 3·12 base pair, we examined the effects of combinations of A²⁰ and helix2 mutants on the suppression of 21GU mutations (Table IV, bottom). The g³·C¹² substitution strongly enhanced the activity of the 21GU/20AC and 21GU/20AU ribozymes but had little effect on the inactive 21GU/20AG ribozyme. The effect of the 3·12 base pair mutation is independent of the nucleotide present at position 20 except for the inactive 20AG mutation (see below). The rescue by a³·U¹² substitution may in some cases obscure the A²⁰ effect.

Two further results point to the likely importance of noncanonical base pairing between A²⁰·C⁴⁴ and G²¹·A⁴³. First, mutations that would be expected to extend Watson-Crick base pairing of helix 3 into loop B are severely inhibitory (Table IV, top). Second, the loss of activity induced by the 20AG transition can be partially rescued by the 44CU mutation (data not shown). A G²⁰·U⁴⁴ wobble pairing is nearly isosteric to the one seen for A²⁰·C⁴⁴ in the NMR-derived structure (22).

The suppressive effect of 20AC is likely to be a consequence of a local conformational effect within loop B; this could, for example, reflect the presence of a Mg²⁺ binding site in between A²⁰ and G²¹ (26).

Effects of A⁴³ Mutations on the Suppression of G²¹ Mutants-- In the model of loop B structure, G²¹ forms a sheared base pair with A⁴³ (16, 20, 22, 49). Therefore, the functional effects of A⁴³ mutations were examined in the wild type molecule (Table V). Since G²¹ is also linked genetically to the 3·12 base pair, we also examined the effects of base pair changes at 3·12 on the A⁴³ mutants. The simultaneous mutation of G²¹ and A⁴³ is very detrimental in any context (Table V). The effect of the mutations of the 3·12 base pair is only seen with G⁴³ and is really significant for the g³·C¹² substitution, which partially restores activity in the 21GU/43AG and 21GC/43AG contexts (at least a 40-fold improvement). Together with the data on G²¹, this strongly suggests that the purine requirement at position 43 is not only constrained by the G·A sheared base pair.

UV-Cross-linking Assays to Monitor Folding of Loop B-- The structure-induced photo-cross-linking of G²¹ to U⁴² (20) can be used as an activity-independent assay to monitor folding of the RNA within loop B. We examined the time course and reaction sites of photo-cross-linking for several of the mutants that are under investigation in this study.

Interestingly, all variants at position 21 retain their ability to form a UV-induced cross-link (Fig. 5A), although the yield of cross-linked product is significantly reduced relative to the wild type molecule (Fig. 5B). Mapping of the 5' cross-linking site for the 21GU mutant shows that the cross-link occurs at or in the immediate vicinity of U²¹ (data not shown). The 3' cross-linking site maps unambiguously to U⁴², the same site used by the wild type molecule (note that in Ref. 20 the 3' cross-linking site was incorrectly interpreted as U⁴¹). Together, these results indicate that we observe the same cross-link, reflecting the same local conformation present in all of the molecules tested, although a smaller proportion of the mutant molecules are folded into the photoreactive structure.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   UV-cross-linking analysis of mutants. A, products of cross-linking reactions analyzed on a denaturing polyacrylamide gel. Reactions were carried out as described under "Materials and Methods." Note that the G21 mutants have a 2-base pair elongated helix 4 and therefore have a slightly reduced mobility. B, cross-linking kinetics of variants. F represents the fraction of uncross-linked RNA.

In contrast, A⁴³ mutants show a slightly different pattern. Cross-links to the correct sites are observed for each mutant but with different efficiencies (Fig. 5B). 43AG and 43AU mutants retain a significant amount of cross-linking, while 43AC almost abolishes the photoreactivity.

Although the helix 2 base pair substitution g³·C¹² rescues the cleavage deficiency of the 21GU mutant, it has no effect on the reduction in cross-linking efficiency induced by the same mutation. As expected, the 3ag·12UC substitution has no effect on the cross-linking rate of the wild type molecules.

These results indicate that the photoreactive structure containing cross-strand stacking is robust enough to withstand base substitutions that result in severe loss of catalytic activity. The observation that the g³·C¹² substitution partially rescues activity without increasing the cross-linking efficiency confirms that the helix 2 substitution does not affect folding of loop B.

A Complex between the Substrate and Domain B-- Gel mobility shift assays were used to examine possible interactions between the substrate (S; from u⁵ to u⁺⁹), substrate-binding strand (SBS; from A¹ to A¹³), and domain B (from A¹⁵ to A⁵⁰). When labeled substrate-binding strand was used, the addition of substrate caused a decrease in mobility consistent with formation of the substrate-substrate-binding strand duplex (Fig. 6A). However, no such shift was observed in the presence of domain B, whether or not the substrate was present. These results are consistent with previously published observations, which demonstrate formation of a stable substrate-strand binding site duplex (40, 50, 51). In contrast, a complex was observed when labeled substrate was incubated with domain B (Fig. 6B). Two bands with mobilities lower than that of unbound substrate were observed, with the lower mobility species predominating at higher loop B domain concentrations. These two species may reflect differences in conformation or differences in composition. The titration experiment indicates that the substrate-domain B complex has an apparent K_D of approximately 0.6 µM (Fig. 6B). To better evaluate the significance of the complex, we repeated the experiment with the "docking" mutant +1ga substrate. Our results are fully consistent with what has been observed with the complete ribozyme using fluorescence resonance energy transfer experiments: the +1ga substrate shows a decreased affinity (apparent K_D of 3.5 µM) for the loop B (18).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Binding of substrate to domain B of the hairpin ribozyme. A, nondenaturing gel analysis of labeled substrate-binding strand (SBS), in combination with substrate (S) and domain B. Respective concentrations were 1 nM substrate-binding strand, 100 nM substrate, and 5 mM loop B. No complex between substrate-binding strand and loop B was observed with concentration in loop B up to 30 mM. Note that in the lane containing the three pieces of RNA, no substrate-binding strand-substrate complex is detected because the substrate is titrated by the loop B (see Fig. 6B). For detailed experimental procedures, see "Materials and Methods." B, nondenaturing gel analysis of complex formation between labeled substrate and unlabeled domain B. Experiments are described under "Materials and Methods." C, nondenaturing gel pattern of a binding assay using 21GU domain B, in combination with wild-type substrate and with 3ag variant substrate. D, formation of the complex between loop B and the substrate, as a function of loop B concentration. , WT substrate and WT loop B. , WT substrate and 21GU loop B. , 3ag substrate and 21GU loop B.

To examine the possibility of fortuitous base pairing between the substrate and the loop B domain, we used a DNA analog of the substrate as a control and found that no complex could be detected (data not shown). We tested the effect of combinations of mutations at ribozyme position 21 and substrate position 3. Strikingly, the 21GU substitution in domain B eliminated the formation of the substrate-domain B complex, while the 3ag substrate substitution restored complex formation (Fig. 6, C and D). The apparent dissociation constant for the complex between the mutant substrate and mutant domain B is 0.1 µM, slightly lower than that of the wild type molecules (Fig. 6, C and D). It also runs more homogeneously than the wild type, consistent with the slight increase of activity (Fig. 6C). Disruption and restoration of the substrate-domain B interaction by the same mutations that inhibit and rescue catalytic activity suggest that this complex could be relevant to normal ribozyme function. However, it is important to note that we have made numerous attempts to identify catalytic activity in the substrate-domain B complex under a variety of conditions, and no evidence for catalytic function has been obtained.

	DISCUSSION

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Isolation of Suppressor Mutations by in Vitro Selection-- In vitro selection technology has provided new tools for investigating the structure-function relationships of biologically important nucleic acids and for the isolation of RNA molecules with novel properties from random sequence pools. Here, we report an in vitro selection strategy to search for second-site revertants of strongly inactivating point mutations. This strategy has been adapted from organismal genetics, where both intragenic and extragenic suppressors have long been invaluable for identifying functional interactions between sites within and between gene products. Our results show clearly that the same strategy can be successfully conducted entirely in vitro. Biochemical studies of synthetic trans-acting ribozymes confirm that the base substitutions isolated in the selection experiments do, indeed, result in the restoration of RNA cleavage activity to the G²¹ mutant ribozymes. While the reversion of the G²¹ mutant phenotype is clearly established, these studies do not themselves tell us the exact nature of the functional compensation of G²¹ mutations (discussed below).

Structural Importance of the G²¹·A⁴³ Sheared Base Pair-- Because mutations at G²¹ retain some activity, the presence of the G²¹·A⁴³ sheared base pair in the catalytic structure is still a matter of discussion (37, 45). Below we discuss the possibility for the local geometry to withstand G²¹ mutations. The G²¹·A⁴³ sheared base pair exposes N-7 and O-6 of G²¹ on the outside of the helix (22-24, 52-54). Stacking of G²¹ and U⁴² is likely to be responsible for the observed photo-cross-linking reaction (22). In the phylogenetically derived and NMR-derived model, this base pairing appears as a keystone of the peculiar loop B structure. Strikingly, our findings show that some G²¹ and A⁴³ mutations retain a significant cross-linking activity and, even more surprising, that the hairpin ribozyme can recover a full activity despite the presence of this expected important structural defect.

Several lines of evidence suggest that the overall geometry of a G·A sheared base pair may be retained in the presence of mutations. First, the G·A pair contains two hydrogen bonds to the backbone (23, 24), and the geometry of the pair may be maintained with only one base-specific hydrogen bond when a third strand interacts with the sheared pair (53, 54). Second, the base pairs that flank G·A pairs are known to constrain backbone orientation and stacking (55). Finally, hydrogen bonds are formed with the adjacent backbone (54, 56). Congruent with our finding that some mutations can be tolerated, functional group substitutions of a G·A pair in two different sequence contexts can be introduced without dramatic loss of stability (52, 57). Similarly, the two A nucleotides participating in the sheared base pairs of the hammerhead ribozyme can also be substituted with ribopurine without dramatic activity loss (58, 59). These data together with the retention of a substantial photosensitivity suggest that a significant fraction of the mutant molecules still adopt the correct overall geometry. A recent study combining phylogenetic comparison and molecular modeling of the 5 S rRNA loops shows that all the mutations of the G of a G·A sheared base pair can be accommodated with a similar geometry. Our results are in good agreement with Leontis and Westhof modeling, except for the G²¹·U⁴³ and G²¹·G⁴³ mutants that retain some photoreactivity and for which they could not model an isosteric pair (60). Interestingly, the U⁴³ mutant is as photoreactive as the G⁴³ mutant but in contrast is completely inactive and not rescued by the 3ag/12UC substitution; this suggests another role for A⁴³.

In conclusion, it is likely that most of the G²¹·A⁴³ mutants are not so much altered in the overall structure of loop B but rather in another function, which could be the interdomain interaction.

Possible Significance of the Interaction between the Loop B Domain and the Isolated Substrate-- Results of the gel mobility shift experiments demonstrate the formation of a complex between the substrate and domain B, with a dissociation constant of 0.6 µM and sufficient stability to be visualized by electrophoretic methods. No complexes between loop B and the substrate binding strand could be observed, either in the presence or absence of substrate. Particularly striking is the finding that formation of the complex is inhibited by the 21GU substitution and rescued by the 3ag change, in correlation with the loss of cleavage activity that results from 21GU and its restoration by the 3ag change. These results suggest that the mechanism through which 21GU inhibits cleavage activity could involve destabilization of an interaction between domain B and substrate.

The nature of the complex between substrate and domain B is unknown. Examination of the sequences for potential Watson-Crick base pairing revealed the potential to form several short duplexes. The longest of them consists of five contiguous base pairs between the 3'-end of the substrate (u⁺⁵ through u⁺⁹) and loop B positions A²² through A²⁶. However, four observations indicate that these potential short duplexes may not be responsible for the slowly migrating complex. First, no complex was observed when a DNA analog of the substrate was used. Second, modification of the sequence of helices 3 and 4 did not eliminate complex formation. Third, none of the identified potential interactions would be directly affected by the 21GU or the 3cg substitutions. Fourth, Pinard et al. (27) recently demonstrated that the g⁺¹ position of the substrate interacts with C²⁵ of loop B; consistent with this finding, we found that +1ga mutation destabilizes the observed complex.

The complex between substrate and domain B has no detectable cleavage activity and is therefore unlikely to represent an intermediate on the normal cleavage pathway. However, there are several observations suggesting that the physical interaction that we observe may have some relevance to the catalytic structure. First, formation of the complex is disrupted and restored by the same mutations in the two domains that have been identified as inactivating and compensating mutations on the basis of activity-based in vitro selection experiments. Second, covalent cross-linking experiments indicate that hairpin ribozymes derivatized with an azidophenacyl group at the A²⁰-G²¹ linkage form specific adducts with the nucleotides at positions 11 and 12, the latter being the base pairing partner of nucleotide 3 (61). Third, a preliminary structure model of the hairpin ribozyme-substrate complex aligns the two domains such that the 3·12 base pair is closely opposed to the G²¹·A⁴³ pair (16). Finally, hydroxyl radical footprinting shows that the ribozyme portion of helix 2 and A⁴³ are internalized upon docking of the two domains (19).

Nature of the Functional Compensation and Role of the G²¹·A⁴³ Sheared Base Pair-- The results summarized above indicate that G²¹ is important, but not essential, in maintaining the active configuration of loop B. The g³·C¹² substitution renders the activity relatively independent of the nucleotide present at the position 21. Despite the lack of an obvious specificity, this substitution cannot be considered as a trivial general up-mutation, since it does not have a significant effect on most A⁴³ mutants and cannot restore the phenotype of the G⁸,⁵ g⁺¹, and G¹¹ mutants (data not shown). The increase of the wild type molecule upon the g³·C¹² substitution could be due to an improvement in loop A-loop B interaction or simply to the stabilization of a weak helix 2. We do not favor the latter hypothesis, since the rate-limiting step of the hairpin ribozyme seems to be a structural rearrangement upon cleavage or the chemical reaction itself (discussed in Ref. 62). Furthermore, the reverse base pair (c³·G¹²) does not have such a drastic effect.

When run on a nondenaturing gel, the 21GU mutant runs mainly as undocked molecules. Furthermore, our results suggest that both the G²¹·A⁴³ and the 3·12 base pair are involved in interdomain interactions. Finally, we report here the existence of a loop B-substrate complex that is disrupted upon G²¹ mutation and restored by the 3ag substitution. Although we cannot make conclusions regarding the nature and the functional role of this complex (see above), it clearly indicates that some interactions mediated by G²¹ and c³ can be made between the substrate and the loop B domain. This led us to the conclusion that G²¹ variants are "docking" mutants that can be restored by high Mg²⁺ concentration. The lack of interpretable specificity of the mutation suppression pattern does not argue in favor of a direct contact between the G²¹ and the 3·12 base pairs. In contrast, the identity of A⁴³ seems constrained beyond the requirement for a sheared base pair. Furthermore, two lines of evidence suggest that A⁴³ and U¹² are implicated in interdomain interactions. First, using NAIM, Ryder and Strobel (45) identified the Rp phosphate oxygen at A⁴³ and the 2'-hydroxyl at U¹² to be required for optimal activity (45). Second, these two positions are internalized upon docking of the two domains as monitored by hydroxyradical footprinting. We propose that the A⁴³ shallow groove face interacts with the a³·U¹² minor groove. In the most straightforward scenario, A⁴³ interacts with a³·U¹² through a Mg²⁺ ion, which would be superfluous in the presence of g³·C¹². Nevertheless, this hypothesis does not take into account the newly identified metal binding site between A²⁰ and G²¹. We are therefore proposing that the interdomain interaction is sensitive to the positioning of A⁴³ by the G²¹·A⁴³ base pairing, which in turn is likely to be influenced by the binding of a Mg²⁺ ion (see below). G²¹ mutations may alter the configuration of A⁴³, such as it that can no longer interact efficiently with a³·U¹². The substitution 3ag/12UC restores the interaction by providing a new dispatch of interacting functional group in the shallow groove and the additional g³ exocyclic amine. In any construct, this interaction may need a structural rearrangement to make A⁴³ more accessible (reviewed in Ref. 2). Although to date we have been unable to develop a compelling structural model for any of these contacts, our hypothesis seem to be a reasonable explanation for our results and the data accumulated by other authors (19, 45, 47, 63). Such a contact could be incorporated in the model of Earnshaw et al. (16) with minor rearrangements.⁶ Our data cannot rule out that a³·U¹² and G²¹·A⁴³ base pairs are independently involved in two functionally redundant interdomain contacts, although in this case, we would have expect the G²¹·A⁴³ partner to appear in the selection experiment. The existence of an ion binding site at G²¹ is already supported by two independent sets of experiments. First, in previous results of chemical modification experiments, G²¹ shows protection from kethoxal (N-1, N-2) and NiCr (N-7) modification upon folding Mg²⁺ addition (49). Second, Butcher et al. (26) have recently identified a Mg²⁺ binding site between A²⁰ and G²¹ phosphates. In their model, the ion bridges the 2-phosphate and is therefore likely to constrain the geometry of the backbone (see, for example, Ref. 64). Interestingly enough, we isolated 20AC as a suppressor of G²¹ mutations. In this case, it seems likely that a local conformation effect partially restores the Mg²⁺ binding site altered by G²¹ mutations.

The presence of one or two Mg²⁺ binding sites in the hairpin ribozyme is still a matter of discussion. Walter et al. (65) hypothesize that two ion binding sites were necessary for a ribozyme constituted of a four-way junction, while only one was required for the two-way junction ribozyme (65). A careful examination of the sequences used in the different studies showed that the Mg²⁺ requirement is correlated with the identity of the 3·12 base pair independently of the nature of the junction (17, 18, 37, 38, 47, 62, 66). Interpreted in the light of the works cited above, our data show that the WT ribozyme has a requirement for two Mg²⁺ ions, with the g³·C¹² mutation exempting the ribozyme from binding the second ion. Our results strongly suggest that the second Mg²⁺ is bound by A²⁰-G²¹ and that the g³·C¹² mutation allows the ribozyme to make an interdomain contact without having to bind the second Mg²⁺.

It is significant to note that the 3ag/12UC substitutions appear to increase the activity of the wild type ribozyme in low Mg²⁺. In addition, it is present in the majority of the molecules derived through in vitro selection (43) as well as in the two other known natural occurrences of the ribozyme (67, 68). The reason for the presence of a functional but suboptimal base pair in the sTRSV version of the hairpin ribozyme is not known, and it could be imposed by other viral functions.

	ACKNOWLEDGEMENTS

We thank the members of the Burke laboratory for helpful discussions, M. Millham for T7 RNA polymerase preparation, D. B. Pecchia for oligonucleotide synthesis, E. Westhof for fruitful discussions, and S. Butcher for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by the CNRS, a grant from Le Ministère de la recherche Francaise, and research grants from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 33-1-69-82-31-54; Fax: 33-1-69-82-43-86; E-mail: sargueil@smigiris.cgm.cnrs-gif.fr.

Published, JBC Papers in Press, July 20, 2000, DOI 10.1074/jbc.M005591200

¹ B. Sargueil, S. Butcher, and J. Burke, unpublished results.

² S. Butcher, personal communication.

⁴ B. Sargueil, A. Berzal-Herranz, and J. Burke, unpublished results.

⁵ B. Sargueil and J. Burke, manuscript in preparation.

⁶ E. Westhof, personal communication.

	ABBREVIATIONS

The abbreviation used is: WT, wild type.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES