Rules for RNA recognition of GNRA tetraloops deduced by invitro selection: comparison with invivo evolution
1997; Springer Nature; Volume: 16; Issue: 11 Linguagem: Inglês
10.1093/emboj/16.11.3289
ISSN1460-2075
Autores Tópico(s)Viral Infections and Immunology Research
ResumoArticle1 June 1997free access Rules for RNA recognition of GNRA tetraloops deduced by in vitro selection: comparison with in vivo evolution Maria Costa Corresponding Author Maria Costa Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author François Michel Corresponding Author François Michel Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author Maria Costa Corresponding Author Maria Costa Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author François Michel Corresponding Author François Michel Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author Author Information Maria Costa 1 and François Michel 1 1Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France *E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (1997)16:3289-3302https://doi.org/10.1093/emboj/16.11.3289 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Terminal loops with a GNRA consensus sequence are a prominent feature of large self-assembling RNA molecules. In order to investigate tertiary interactions involving GNRA loops, we have devised an in vitro selection system derived from a group I ribozyme. Two selections, destined to isolate RNA sequences that would recognize two of the most widespread loops (GUGA and GAAA), yielded variants of previously identified receptors for those loops, and also some yet unrecognized, high-affinity binders with novel specificities towards members of the GNRA family. By taking advantage of available crystal structures, we have attempted to rationalize these results in terms of RNA–RNA contacts and to expose some of the structural principles that govern GNRA loop-mediated tertiary interactions; the role of loop nucleotide 2 in ensuring specific recognition by receptors is emphasized. More generally, comparison of the products of in vitro and natural selection is shown to provide insights into the mechanisms underlying the in vivo evolution of self-assembling RNA molecules. Introduction Like protein enzymes, large RNA catalysts, such as group I and group II self-splicing introns or the RNA component of bacterial RNase P, fold into compact structures that are stabilized by a multiplicity of tertiary interactions (Latham and Cech, 1989; Cate et al., 1996a). At least some of these interactions must correspond to recurrent structural motifs, whose identification and characterization should be essential to our understanding of the principles underlying RNA folding and catalysis and for future predictions of three-dimensional structures from RNA sequence. That natural, self-assembling RNA molecules tend to make intensive use of a relatively small number of building blocks is suggested indeed by the state of our knowledge concerning terminal loops and their interactions. The sizes and sequences of terminal loops are extremely biased in large natural RNAs with a stable three-dimensional structure; loops with four nucleotides and a GNRA consensus sequence (R stands for a purine and N for any base) may constitute up to one third of the total in some molecules (e.g. Woese et al., 1990). A first indication that GNRA loops frequently participate in RNA tertiary interactions came from comparative sequence analyses of group I self-splicing introns (Michel and Westhof, 1990). These analyses revealed two cases of phylogenetic covariation in which a GUAA loop and a C:G pair in a distant helix had exchanged repeatedly during evolution with the combination of a GUGA loop and a U:A pair. These covariations were proposed to result from a direct contact between the third base of the loop and the shallow ('minor') groove side of the base pair. Since then, additional instances of the same type of covariation have been found in bacterial RNase P RNA (Brown et al., 1996), ribosomal RNA (Gutell, 1996) and group II self-splicing introns (Costa et al., 1997). Moreover, all the interactions proposed to exist in self-splicing introns have been checked to be compatible with biochemical evidence (Jaeger et al., 1993, 1994; Murphy and Cech, 1994; Costa and Michel, 1995; Chanfreau and Jacquier, 1996; Costa et al., 1997). Our first atomic resolution picture of a GNRA loop interacting with RNA was brought by a packing contact between two consecutive C:G pairs and a GAAA loop in crystals of the hammerhead ribozyme (Pley et al., 1994). In addition to confirming that GNRA loops dock into the shallow groove of RNA helices, this structure revealed a network of hydrogen bonded contacts involving 2′ hydroxyl groups on both the loop and loop receptor sides. More recently, the crystal structure of a 160 nucleotide (nt) domain from a group I intron (Cate et al., 1996a) has provided us with one instance of an intramolecular contact involving a GAAA loop. The receptor in that case does not consist of two C:G pairs, but of an 11 nt motif (CCUAAG..UAUGG) that had already been shown by Murphy and Cech (1994) and Costa and Michel (1995) to interact with GAAA loops in self-splicing introns (this motif is also present in the RNase P RNA of some Gram-positive bacteria; Tanner and Cech, 1995). Despite these advances, our understanding of tertiary interactions involving GNRA loops remains fragmentary. Partners have not yet been identified for a majority of GNRA loops in large natural RNAs. Although a number of these loops will probably turn out to be recognized by proteins (e.g. Glück et al., 1992), others, especially in self-assembling molecules, must be contacted by yet unidentified RNA receptors. Neither has there been any comprehensive investigation into the specificity of currently known receptors towards the various members of the GNRA family. Published phylogenetic and biochemical evidence could be taken to argue that the smaller receptors, which seemingly consist of only two base pairs, are poor discriminators, while the (CCUAAG..UAUGG) sequence, which is so frequent in self-splicing introns, is highly specific for GAAA loops (Costa and Michel, 1995). However, specific partners are likely to exist for other GNRA loops as well. The question then is whether these motifs are also used by nature, and if not, why not? We reasoned that in vitro selection of RNA motifs capable of recognizing GNRA loops would not only constitute the most powerful strategy to recover any missing receptors for these loops, but should also greatly help our understanding of the structural principles that govern these tertiary interactions. Accordingly, we have devised an in vitro selection system suitable for the isolation of RNA motifs that specifically bind terminal loops and we have used this system, which is based on mutual recognition of a group I ribozyme and its substrate, to look for molecules that would recognize the GUGA and GAAA loops. After seven rounds of selection and amplification, variants of previously identified receptor motifs were found to predominate among selected molecules. However, both final pools also contain a number of new receptor sequences. These sequences are in no way inferior to previously identified motifs in terms of efficiency of recognition, but show novel patterns of discrimination, especially between loops that differ from one another by their second nucleotide. Rationalization of these results with the help of available structural data leads to general principles regarding the interaction of GNRA loops with their receptors and provides some insights into the mechanisms that underlie the evolution of loop–receptor partnerships in nature. Results In vitro selection of GUGA- and GAAA-binding motifs Our in vitro selection system is based on the td molecule, a group I intron that interrupts the thymidylate synthase gene of bacteriophage T4 (Belfort et al., 1987). We have shown previously that in the wild-type intron, the GUGA loop at the tip of helix P2 specifically binds the CU:AG sequence of helix P8, at positions four and five when counting from the base of the latter helix (Figure 1A; Costa and Michel, 1995). The td intron was transformed into a bimolecular system (Figures 1 and 2) by splitting a molecule composed of the intron itself and a short 5′ exon into a 'substrate', formed by hairpin structures P1 and P2, and a 'core', which comprises the rest of the intron (see Materials and methods). We verified that co-incubation of these two pieces in the absence of the guanosine cofactor of group I self-splicing is followed by attack of the normal 5′ splice site within the P1 helix by the terminal G residue of the intron, resulting in a new covalent bond between the two ends of the intron sequence. This addition reaction, which rests on the ability of the core to recognize and properly position the substrate into its active site, forms the basis for our selection procedure, which is shown in Figure 2 and is very similar to the one used by Robertson and Joyce (1990). 'Chimeric' core–substrate products are reverse-transcribed with an oligonucleotide primer designed to ensure selective recovery of those core molecules having catalyzed nucleophilic attack at the proper 5′ splice site. The next step consists of PCR amplification of cDNA molecules with a set of oligonucleotides that introduces a T7 promoter and deletes all remaining substrate nucleotides from the core. T7 transcription of the resulting DNA matrices yields a new population of core molecules ready for another round of selection. Figure 1.Conversion of the td intron of bacteriophage T4 into a two–piece system to allow in vitro selection of co-adapted L2 and P8 sequences. (A) Our original td construct. The arrow points to the 5′ splice site. The td 5′ exon was replaced by a 14 nt sequence, the last seven bases of which belong to the original td sequence. Dashed lines indicate the interaction between the GUGA L2 loop and its receptor in the P8 helix. Note that the UGAG sequence at the P1–P2 junction can pair with either the sequence to its left (5′ of P1) or the one to its right (3′ of P2). (B) Constructs used for in vitro selection. The sequence of the P1–P2 'substrate' piece is that shown in (A): only the sequence of the L2 loop differed from one substrate to the next. The second piece was derived from the catalytic core of the intron, beginning immediately 3′ of the P1–P2 sequence and ending with the intron 3′ terminal G (a G was added at the 5′ end of this piece, to allow efficient transcription; see also Costa and Michel, 1995). The distal section of the P8 hairpin structure was replaced by a random sequence of 21 nucleotides. Core molecules are selected for their ability to interact with the L2 loop of a P1–P2 substrate. Download figure Download PowerPoint Figure 2.In vitro selection procedure. The horizontal arrow above the U°G wobble pair of the P1–P2 substrate marks the site of attack by the intron terminal G (circled), which is shown sitting in the group I guanosine-binding site (symbolized by a dark grey sector). Binding of the substrate by the intron core is dependent on recognition of the L2 loop by an appropriate P8 sequence. An oligonucleotide complementary to the distal part of P1 and the last nucleotides of the intron (see Materials and methods) allows selective reverse transcription of reacted core molecules carrying a correct core–substrate junction. The same initial population of core molecules was used to carry out two selection experiments with substrates that differed only by the sequence of their L2 loop (GUGA or GAAA). See Materials and methods for experimental details. Download figure Download PowerPoint Preliminary experiments (not shown) allowed us to verify that just like attack by free guanosine (Costa and Michel, 1995), the efficiency of the addition step depends strongly on the nature of the L2 and P8 partners. Using a wild-type td core, the addition reaction was optimal with a P1–P2 substrate carrying a wild-type, GUGA L2 loop, much poorer with a GAAA loop, and barely detectable when the loop was UUCG (UUCG loops do not interact with known receptors for GNRA loops; Jaeger et al., 1994; Murphy and Cech, 1994). We then constructed a population of intron core molecules (see Materials and methods) in which 16 of the 20 nucleotides of the original P8 hairpin of intron td had been replaced by a random sequence of 21 bases (Figure 1B). Five extra positions were added to the wild-type structure in order to avoid missing potential binding motifs larger than the wild-type one. On the other hand, the first 2 bp of P8 were left unchanged: we reasoned that maintaining base pairing at the base of P8 would further the formation of hairpin-like structures and reduce the risk of disturbing the overall architecture of the core. An additional reason for leaving the nucleotides at the base of P8 unaltered is that these residues tend to be well-conserved among relatives of the td intron (members of subgroups IA and IB; see Michel and Westhof, 1990) and may therefore be involved in tertiary contacts. In contrast, there is a complete lack of sequence conservation in the rest of P8 in those members of subgroups IA and IB which lack a P2 stem (Michel and Westhof, 1990): this observation, which strongly suggests that the section of P8 that was randomized, has no other function in td and related introns other than binding the L2 loop, vindicates the choice of P8 as a target for the type of selection that was carried out in this work. With a mass of 7.5 pmol, our initial pool of core molecules must have contained some 64% [1−(1−4−21)n, with n = 7.5×10−12×6.023×1023] of all possible P8 sequences of 21 nucleotides. The same initial population of core molecules was used to carry out two selections in parallel: one for binding of the GUGA loop and the other one for binding of the GAAA loop. For each experiment, seven rounds of selection and amplification were performed, resulting in two final selected pools, designated as the 'GUGA' and 'GAAA' pools. Figure 3 shows the products of addition reactions of the td core, of the initial pool and of the final, selected pools. In each case, a major reaction product whose existence depends on the presence of a substrate molecule can be seen: its electrophoretic mobility is the one expected for a molecule consisting of the intron core and the 3′ portion of the substrate. Comparison of the addition reactions of the initial pool (Figure 4A) with those of the final pools (Figure 4B) is indicative of the degree of improvement achieved by selection (note that the conditions used in Figure 4B, which are those of the last round of selection, are more stringent than the ones of Figure 4A, which correspond to the first round). Moreover, adaption was specific, since final pools were found to react much better with the substrate with which they had been confronted (Figure 4B). Interestingly, reaction of the td core with the wild-type (GUGA) substrate was significantly slower than that of the final GUGA pool, betraying the fact that the former molecule is not optimal in terms of substrate binding (see below). However, an even more efficient reaction was obtained for the combination of the final GAAA pool and GAAA substrate. Figure 3.Addition reaction products of the initial and final pools. Core molecules were incubated with a substrate carrying a GUGA or GAAA loop for either 20 min (initial pool) or 10 min (final GUGA and GAAA pools and wild-type td core); control reactions with no substrate or no core were included; see legend to Figure 4 for reaction conditions. Products were identified from their electrophoretic mobilities (expected sizes were 235 nt for core molecules of the various pools and 230 nt for the wild-type td core, with 36 additional nucleotides for addition products). Only the top and bottom of a 6% denaturing polyacrylamide gel are shown (no products were detected in the rest of the gel). The bottom part of the gel was less exposed than the top in order to compensate for the higher specific activity of the substrates (substrate over core labelling ratios ranged from 32 to 34.8). Download figure Download PowerPoint Figure 4.Kinetics of addition reactions of P1–P2 substrates with intron core molecules. Filled symbols, GUGA substrate; empty symbols, GAAA substrate. (A) Time course of addition reactions resulting from incubation of the initial pool with each one of the substrates under the reaction conditions used for the first round of selection (45°C, 50 mM magnesium buffer, 4 μM substrate, 0.4 μM core molecules). (B) Time course of addition reactions of the final GUGA (circles) and GAAA (diamonds) pools and of the wild-type td core (crosses); the latter was incubated only with the wild-type, GUGA substrate. Reaction conditions were as in (A), except that the buffer contained 20 mM magnesium. Download figure Download PowerPoint Because of competition between addition and hydrolysis at the core–substrate junction, all reactions in Figure 4B eventually level off. Importantly, by using chimeric core–substrate molecules carrying previously characterized, matched and mismatched L2×P8 combinations (Costa and Michel, 1995), we checked (data not shown) that hydrolysis is indeed specific to the 3′ end of the intron and that its rate is largely insensitive to the strength of the L2×P8 interaction. Therefore, hydrolysis does not antagonize selection for binding efficiency. Several families of receptors for GUGA and GAAA tetraloops We sequenced the entire intron core of some 30 individuals from each of the final pools. As expected from our use of a high-fidelity DNA polymerase for all amplifications, few molecules were found to carry mutations outside the randomized P8 segment. Moreover, the positions affected were never the same, except for a recurrent G to A mutation at the first position of the J8/7 segment, immediately 3′ of P8. This mutation is present in 18% of clones from the final GUGA pool (Figure 5A) and its role in the recognition of substrate molecules by the core will be described elsewhere. These data are consistent with the view that the L2×P8 interaction was the main target for improvement of substrate binding. Figure 5.Sequence alignment of the P8 domains of selected core molecules. The segment of sequence that was randomized is shown in bold type: for each class, one representative sequence is shown in full, with identical nucleotides being replaced by dots in the other sequences. Potential secondary structure pairings are indicated by dashes and divergent arrows below a sequence. Vertical arrows point to the C:G base pair that is common to most currently known receptors and was found to interact with the last A of GNRA loops in crystal structures (see text). Residues in lower case stand for dinucleotides of the corresponding residue. Roman numerals and letters designate classes and subclasses of P8 sequences (see Results). Code names in bold type are those of the clones that were characterized kinetically. (A) Alignment of 33 pool GUGA sequences with the wild-type T4.td sequence. In classes IB and II, a gap was introduced between the first and second nucleotides of P8 to force alignment of the receptor sequence with that of td (a number of natural group I sequences have a gap at the same position; see Michel and Westhof, 1990). A recurrent G to A mutation of the first nucleotide of the J8/7 segment is marked by an asterisk (see Results). (B) Alignment of 35 clones belonging to the final GAAA pool. Download figure Download PowerPoint Alignment of the P8 segments of selected individuals (Figure 5) revealed the presence of several classes of receptor for each loop. In the GUGA pool, up to five receptor families—defined on the basis of the sequence at positions four and five of P8—may be distinguished (Figure 5A). Much as expected, the most abundant family is the one with a CU:AG sequence: CU:AG helices were shown previously to be specific receptors for GUGA loops (Michel and Westhof, 1990; Jaeger et al., 1994). Importantly, this sequence is most often located at the same place as in the td intron, which confirms that splitting the td molecule into two transcripts does not alter the relative positioning of the P2 and P8 helices. In fact, most sequences may be aligned in such a way that they share either base pair 4 or 5 with the td sequence. However, class IV molecules, which have a G[N0–4]GCU:GGCC consensus sequence, appear unrelated to either the td molecule or other clones. Finally, the observation that 11 of the 16 clones in subclasses IA to IC have a C:G pair on the distal side of their CU:AG receptor motif suggests that this position could also be involved in some kind of contact with the GUGA loop. Two families of receptors were recovered from the GAAA pool (Figure 5B). Aside from the five class II molecules, in which a CCC:GGG consensus sequence from positions 3 to 5 is followed by an asymmetric internal loop, the majority of the clones harbour either a canonical version or variants of an 11 nt motif (CCUAAGUAUGG), which we have shown previously to bind GAAA loops with remarkable affinity (Costa and Michel, 1995). Among minor variations that were selected, the most frequent ones are a C instead of an A at position 5 (see Figure 6 for numbering of the 11 nt receptor), and an A:C combination instead of G:U at the tip of the receptor. However, more divergent variants of the same 11 nt motif were also recovered. In subgroup IB, the UAA sequence of the 5′ branch is replaced by UGY and in three of the four clones, various mismatches substitute for the G:U pair. In subgroup IC, the same UAA sequence becomes UGNA and is most often followed by a C:G pair. Figure 6.Schematic diagram of the structure of the 11 nt receptor and its interactions with a GAAA loop. The figure, which was redrawn from Cate et al. (1996), is meant to evoke the actual crystal structure. Dashed lines stand for hydrogen bonded contacts. The first adenosine of the tetraloop contacts a reverse Hoogsteen U:A pair in the receptor (drawn as U3°A8). The second adenosine recognizes the receptor via 2′ hydroxyl contacts. The third adenosine, which makes a non-canonical base pair with the first nucleotide of the loop, also contacts the C2–G10 pair of the receptor. Nucleotides A4 and A5 are drawn side by side, to indicate that they form a platform by stacking on U7 and G6, respectively (stacking extends in fact from G6 and A5 to the adenines of the loop). U9 bulges out of the middle part of the receptor and contacts A5 (see text). The thin dotted line indicates a 2′ hydroxyl-mediated interaction between the first C:G pair of the receptor and the base pair above the tetraloop. Download figure Download PowerPoint Kinetic characterization of some selected motifs In both final pools, the major class of sequences corresponds to a motif that was already known to be a specific receptor for the loop to which this pool was confronted. Therefore, it is clear that our selection system reproduces at least some of the evolutionary forces that shape receptors in nature. One possible difference, however, between natural conditions and ours is the absence in our case of counterselection for cross-recognition of a receptor by other members of the GNRA family. In order to estimate not only the efficiency of recognition, but also its specificity, we have resorted to kinetic characterization of loop–receptor pairs: selected molecules were assayed for their ability to bind not only their cognate loop but also those among the other members of the GNRA family that differed from it by one nucleotide. Rather than using addition reactions, the analysis of which is complicated by hydrolysis at the junction of the core and substrate, we went back to a reaction that mimics the first step of self-splicing. In the presence of excess guanosine, 3′-truncated intron core molecules act as true catalysts and promote specific cleavage of P1–P2 substrates at the 5′ splice site (Costa and Michel, 1995). Figure 7 shows time courses of cleavage in the presence of excess enzyme for five different L2 substrates incubated with the same ribozyme; all L2×P8 combinations that were tested yielded similar data, compatible with first-order kinetics. Single-turnover reactions were performed at ribozyme concentrations much lower than Km (see Materials and methods) so as to estimate directly the value of kcat/Km (Table I) from rates of reaction. Importantly, all ribozyme–substrate combinations were checked by polyacrylamide gel electrophoresis (data not shown) to cleave at the correct 5′ splice site. Figure 7.Examples of time courses of cleavage reactions of P1–P2 substrates by a td-derived ribozyme. Reactions were carried out under single-turnover and kcat/Km conditions (see Results and Materials and methods) with 0.5 μM wild-type td ribozyme and 0.05 μM substrate (Km was previously determined to be 13.5 μM for the combination of the td ribozyme and a GUGA substrate; see Costa and Michel, 1995). Ordinates are natural logarithms of the unreacted fraction. Plots could be fitted to single exponentials after correction for the presence of at most 12% of inactive substrate molecules. P1–P2 substrates differed only by the sequence of their L2 loop. Symbols: (○), GUGA; (⋄), GCGA; (□), GUAA; (+), GAGA; (▵), GGGA. Download figure Download PowerPoint Table 1. Values of kcat/Km (×105/min/M) for cleavage reactions of P1–P2 substrates by td-derived ribozymes Ribozymes Substrates (L2 loop) GYRA GRRA GUGA GCGA GUAA GCAA GAGA GGGA GAAA GGAA P8 T4.td (CUU:AAG) 1.32 0.89 0.33 ND 0.13 0.048 0.09a ND P8 B7.6 (CUC:GAG) 1.14 0.95 0.26 ND 0.10 0.042 ND ND P8 B7.8 (GCUAC..AGGC) 3.42 0.039 6.97 ND 0.37 0.05 ND ND P8a (CCU:AGG) 0.36a ND 1.03a ND ND ND 0.12a ND P8 11 nt motif (CCUAAG..UAUGG) 0.03a ND 0.38 0.13 0.79 ND 47.5 0.18 P8 C7.2 (CCUGUAC..GAUGG) ND ND 0.27 0.12 0.67 ND 47.5 0.22 P8 7.34 (CCCCACGC..GAAGGG) ND ND 2.86 0.83 2.46 ND 6 17.6 Ribozymes are designated by the name of the clone in Figure 5 and the relevant section of their P8 receptor (P8 bp 4 is in bold type). Values of kcat/Km were determined by dividing first-order rates (determined from the type of data shown in Figure 7) by ribozyme concentrations. ND; not determined. a Data from Costa and Michel (1995). These values, which were determined under the same experimental conditions and had been normalized to the kcat/Km value of the wild-type L2×P8 combination, were multiplied here by the kcat/Km of the td×GUGA combination (1.32). Substrates (L2 loop) GRRA We chose to characterize clones B7.6 and B7.8 from the GUGA pool (Figure 8). Like the majority of class I molecules, clone B7.6 differs from the td intron in that it has a C:G rather than a U:A pair on the distal side of its CU:AG receptor sequence. However, as shown in Table I, neither the affinity of the core for the GUGA substrate (as far as it can be inferred from values of kcat/Km: see Discussion), nor its pattern of discrimination between different GNRA loops is significantly altered by this substitution. On the other hand, the rates measured for clone B7.8, which carries the highly divergent class IV consensus sequence, suggest a different network of molecular contacts. Figure 8.Receptors tested for their ability to discriminate between GNRA loops. The relevant part of the P8 sequence of each one of the ribozymes tested is shown (we checked that these ribozymes differ only by the sequence of their P8 segment). The two base pairs in lower case are the ones that were not randomized. The base pair interacting with the last A of GNRA loops is indicated by an arrow. This pair is C:G in all currently known receptors, with the only exception of the class III and class IV clones of the final GUGA pool, which have a G:C pair at that position (indicated by a question mark in clone B7.8). Download figure Download PowerPoint In the GAAA pool, clone C7.2 was investigated in order to determine to what extent the replacement of UAA by UGNA in the 11 nt receptor motif might interfere with binding of GAAA and the other tetraloops. As can be seen in Table I, the answer is that this substitution has negligible effects on those parts of the receptor that directly contact the tetraloop. In contrast, the C7.34 molecule, which is typical of class II GAAA receptors, shows widely different preferences. Role of the second loop nucleotide The recently determined X-ray structures of two loop–receptor pairs (Pley et al., 1994; Cate et al., 1996a) provide a framework within which sequence and kinetic data may be discussed in terms of specific contacts between GNRA loops and their receptors. Confrontation of biochemical and structural data is especially illuminating in the case of loop nucleotide 2, whose role in loop–receptor recognition had been underestimated. Thus, the receptor for GUGA loops has until now been assumed to consist of only two consecutive base pairs with a CU:AG sequence (Michel and Westhof, 1990; Jaeger et al., 1994). However, in 13 of the 15 clones in subclasses IA and IB of the GUGA pool, helix P8 extends beyond the CU:AG sequence at positions 4 and 5. When the terminal loop that caps the CC:GG receptor of Pley et al. (1994) is replaced by additional Watson–Crick b
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