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A three-dimensional perspective on exon binding by a group II self-splicing intron

2000; Springer Nature; Volume: 19; Issue: 18 Linguagem: Inglês

10.1093/emboj/19.18.5007

1460-2075

María Costa,

Viral Infections and Immunology Research

Article15 September 2000free access A three-dimensional perspective on exon binding by a group II self-splicing intron Maria Costa Maria Costa Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA, 95064 USA Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author François Michel Corresponding Author François Michel Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA, 95064 USA Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author Eric Westhof Eric Westhof Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, France Search for more papers by this author Maria Costa Maria Costa Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA, 95064 USA Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author François Michel Corresponding Author François Michel Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA, 95064 USA Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France Search for more papers by this author Eric Westhof Eric Westhof Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, France Search for more papers by this author Author Information Maria Costa1,2, François Michel 1,2 and Eric Westhof3 1Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA, 95064 USA 2Centre de Génétique Moléculaire du CNRS, 91190 Gif-sur-Yvette, France 3Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5007-5018https://doi.org/10.1093/emboj/19.18.5007 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have used chemical footprinting, kinetic dissection of reactions and comparative sequence analysis to show that in self-splicing introns belonging to subgroup IIB, the sites that bind the 5′ and 3′ exons are connected to one another by tertiary interactions. This unanticipated arrangement, which contrasts with the direct covalent linkage that prevails in the other major subdivision of group II (subgroup IIA), results in a unique three-dimensional architecture for the complex between the exons, their binding sites and intron domain V. A key feature of the modeled complex is the presence of several close contacts between domain V and one of the intron–exon pairings. These contacts, whose existence is supported by hydroxyl radical footprinting, provide a structural framework for the known role of domain V in catalysis and its recently demonstrated involvement in binding of the 5′ exon. Introduction Progress in understanding group II self-splicing has been hindered by our ignorance of how key components of the large group II ribozyme are brought together. For instance, it has long been known that group II introns recognize their 5′ exon by means of two distinct exon-binding sequences, EBS1 and EBS2, that form (typically) six base pairs each with two sequence stretches called IBS1 and IBS2 at the 3′ end of the exon (Jacquier and Michel, 1987). Unfortunately, subsequent studies have mostly failed to provide insight into how the EBS elements, which are located far away in secondary structure models from those sections of the intron that are conserved in sequence, may be connected to the active center of the ribozyme. Only recently could it be demonstrated (Costa and Michel, 1999) that tight binding of the 5′ exon requires not only intron domain I, of which the EBS sequences are part, but also the distal part of domain V, a small yet major component that is believed to be involved in catalysis (Chanfreau and Jacquier, 1994; Peebles et al., 1995; Abramovitz et al., 1996; Konforti et al., 1998a). Even so, it has remained unclear whether domain V contacts the exon, one of the EBS segments or yet another intron component that is itself involved in exon binding. Another source of uncertainty has been the identity of EBS3, the intron-contained binding site for the 3′ exon. It is often regarded as well established that like group I introns, the group II ribozymes guide the ligation of their exons by getting them to bind next to one another to a continuous 'internal guide sequence' (IGS). The fact is that the nucleotide immediately 5′ of EBS1 (δ or EBS3 in Figure 1A) can more often than not form a canonical pair with the first nucleotide (IBS3) of the 3′ exon (Michel and Jacquier, 1987) and experiments have shown that under some conditions cleavage at the 3′ splice site can be redirected by base substitutions at the δ position (Jacquier and Jacquesson-Breuleux, 1991). Moreover, reverse splicing of some group II introns into their DNA target site, which involves pairing of EBS1 and EBS2 with DNA counterparts of IBS1 and IBS2, is significantly more efficient when the δ base can pair with the nucleotide 3′ of IBS1 (Guo et al., 1997; Mohr et al., 2000). However, a potential complication to this picture stems from the existence of two major subdivisions of group II introns (Michel et al., 1989). Among features that differentiate the two subgroups is the relative location of the EBS1 sequence within the ID3 terminal loop (Figure 1). Contrary to subgroup IIA introns, which have at least three unpaired nucleotides 5′ of EBS1, most members of subgroup IIB have only one base available for pairing with the 3′ exon and would seem unlikely to stack it on top of EBS1 without unwinding part of the ID3 helix [compare Figure 1A and B; the helical continuity of the IGS is an integral part of group I and group II guide models and is supported by experiments in which the hydrolytic cleavage reaction catalyzed by a group II intron was shown to strongly prefer extended double-stranded structures with canonical base pairs on both sides of the reactive bond (Jacquier and Jacquesson-Breuleux, 1991; see also Michel and Ferat, 1995; Jacquier, 1996)]. As already noted (Michel et al., 1989), this difference between the two subclasses appears to be reflected in the distribution of bases at the δ and IBS3 sites. Whereas only three out of 58 subgroup IIA introns possess non-matching δ:IBS3 combinations (Figure 1A), thorough examination of subgroup IIB sequences fails to reveal any statistical evidence of base pairing between the δ base and the first residue of the 3′ exon (Figure 1B). Figure 1.Distinct modes of guiding exon ligation in subgroup IIA and IIB introns. (A) Subgroup IIA. Hollow lines and circles indicate stretches of intron sequence and individual intron residues; (---), canonical base pairs; EBS1 and EBS3, exon-binding sites; IBS1 and IBS3, intron-binding sites; ID3, a helix that is part of domain I (see Figure 3); δ is the nucleotide immediately 5′ of EBS1. The table at the bottom shows the distribution of bases at the δ (EBS3) and IBS3 sites in 58 subgroup IIA sequences; mutual information (Chiu and Kolodziejczak, 1991), which measures the extent to which the base contents of one site can be predicted from those of the other one, is high (0.834). The use of EBS3–IBS3, rather than δ–δ′ (Jacquier and Jacquesson-Breuleux, 1991), to designate interactions between the intron and 3′ exon is made necessary by the existence of a distinct δ–δ′ pairing in subgroup IIB molecules (B) and improves the consistency of the group II nomenclature, by restricting Greek letters to tertiary interactions within the intron (Figure 3). (B) Subgroup IIB. Same as (A). Question marks refer to potential interactions, the existence of which is established in this work (see Figure 3). The table at the bottom shows that the base contents of the IBS3 and δ sites are not significantly correlated in 69 subgroup IIB sequences (mutual information 0.127). Download figure Download PowerPoint While it might be concluded from the preceding argument that exon ligation is not generally guided in subgroup IIB introns, we now report that the first residue of the 3′ exon is actually base paired to the intron during exon ligation catalyzed by a self-splicing subgroup IIB molecule. However, the intron partner (EBS3) of the 3′ exon IBS3 site is not the δ nucleotide, which is itself engaged in base pairing with another intron residue (δ′). The two newly identified sites, EBS3 and δ′, happen to be facing each other within an internal RNA loop, the sequence and location of which are well conserved in subgroup IIB introns. We propose that this loop, by means of its specific fold, is primarily responsible for positioning the IBS3–EBS3 pair and EBS1–IBS1 helix in the appropriate conformation for exon ligation. The novel structural constraints uncovered in this work make it possible to build a three-dimensional model of a large section of the active center of group II introns. We further show that this model is supported by data from experiments in which hydroxyl radicals are used for the first time to probe the higher order structure of a group II ribozyme and its complexes with the exons. Results Identification of G293 as a potential exon-binding site In order to uncover a possible binding site for the first base of the 3′ exon, we have chemically probed a group IIB self-splicing intron either alone or in the presence of wild-type and mutated versions of its ligated exons. Group II introns are normally excised as branched molecules called lariats, and the lariat form of intron Pl.LSU/2 from Pylaiella littoralis mitochondria was previously shown to form populations of molecules with a seemingly uniform conformation, which makes it suitable for direct chemical probing (Costa et al., 1997b, 1998; Costa and Michel, 1999). As shown in Figure 2A, probing the Pl.LSU/2 lariat with dimethylsulfate (DMS) in the presence of a 16mer oligoribonucleotide (LE/wt, Table I) that encompasses the last 13 residues of the 5′ exon and the first three nucleotides of the 3′ exon results in the expected footprinting of the A-rich EBS2 sequence and (to a lesser extent) of position A258, within EBS1 (Figure 3; both footprints are attributable to the 5′ exon; see Costa and Michel, 1999). In this case, comparison with another oligonucleotide with a base substitution (C to A) at the first position (+1) of the 3′ exon (LE/C+1A, Table I) revealed no difference that could be ascribed to that substitution. In contrast, when kethoxal, which specifically targets guanines with accessible N1 and N2 groups, is used as a probe, the strong protection that the LE/wt molecule confers to the otherwise (Figure 2A, lane −LE) very reactive G293 is no longer observed if this molecule is replaced by its LE/C+1A counterpart. That this footprint is specific to the 3′ exon was checked further by verifying (Figure 2B) that a previously described (Costa and Michel, 1999) unreactive 13mer analog of the 5′ exon (3′dE5, Table I) completely fails to protect position 293. Figure 2.Mapping by reverse transcription of DMS and kethoxal exon footprints on the lariat form of intron Pl.LSU/2. All modification reactions were at 30°C for 0 (−), 7 (+) or 15 (++) min. C, T, A and G are sequencing lanes (recall that reverse transcriptase stops one nucleotide before a modified base). (A) DMS and kethoxal probing of wild-type lariat either alone (−LE) or in the presence of 2 μM LE/wt or LE/C+1A (Table I) ligated exon molecules. (B) Kethoxal modification of wild-type and C252G intron lariats in the absence or presence of an inactive oligonucleotide analog of the 5′ exon (3′dE5, Table I and Costa and Michel, 1999). Saturation conditions (see text) were ensured by using 6 μM (wild-type lariat) or 30 μM (C252G lariat) 3′dE5. Download figure Download PowerPoint Figure 3.(A) Secondary structure model of the Pl.LSU/2 intron, redrawn from Costa and Michel (1999) with minor modifications (as suggested by comparative analysis, G241 is paired with C276 rather than U279). Individual nucleotides are shown only in those sections of the molecule that are modeled in Figure 6. Greek letters designate tertiary interactions within the intron (Costa and Michel, 1995; Michel and Ferat, 1995; Costa et al., 1997a; Boudvillain and Pyle, 1998; this work). Pairs of helices proposed to stack end-to-end (see text) are indicated by gray bars. Open and filled inverted triangles indicate domain V residues whose reactivity to hydroxyl radicals is increased or decreased, respectively, upon binding of the 5′ exon. Note that numbering takes into account the open reading frame in domain IV, which was removed in the constructs used for in vitro experiments (Costa et al., 1997b). (B) Subdomain ID in intron Aae.LSU/5 from Agrocybe aegerita (DDBJ/EMBL/GenBank accession No. AF087656). Boxed nucleotides are in common with Pl.LSU/2. Other related introns lack bulges in stems ID3(ii) and ID(iv)+ID3(i) (M.Granlund, F.Michel and M.Norgren, submitted; F.Lang, personal communication). (C) Subdomain IC in introns Ppu.LSU/1 from Porphyra purpurea (Burger et al., 1999) and Cal.x1 from Calothrix PCC7601 (Ferat and Michel, 1993). Homologous nucleotides are boxed with thin lines. Heavy boxing: three-way junction specific to the Porphyra intron. Download figure Download PowerPoint Table 1. Oligonucleotides for footprinting experiments and kinetic analysesa Name Sequence rE5 5′-UGUUUAUUAAAAA 3′dE5 5′-UGUUUAUUAAAA3′dAb 2′dE5 5′-UGUUUAUUAAAA2′dAc LE/wt 5′-UGUUUAUUAAAAACAC LE/C+1A 5′-UGUUUAUUAAAAAAAC a Unless otherwise stated, sugars are riboses; the IBS1 sequence (Figure 3) is underlined. b The terminal 3′-OH group is replaced by a hydrogen. c The 2′-OH group of the 3′-terminal nucleotide is replaced by a hydrogen. Disruption of the G293-C+1 pair impairs exon ligation In the experimental set-up we chose to use (Scheme 1), ligated exons (LE) result when the reaction intermediate, a lariat molecule consisting of the branched intron with the 3′ exon still attached (IE3), is incubated with a saturating (Costa and Michel, 1999, and Table II) concentration of the 5′ exon (E5). However, additional products are generated, due to the reversibility of the transesterification reaction that constitutes the first step of the splicing pathway (left part of Scheme 1). Thus, a fraction of IE3·E5 complexes react to reconstitute transiently the precursor (E5IE3). Moreover, the lariat intron (I) produced during exon ligation is also 'debranched' by the 5′ exon to generate E5I, a linear intron–5′ exon molecule [Scheme 2, which can be investigated separately by mixing purified lariat intron molecules with E5, and which was shown by Chin and Pyle (1995) to reach an equilibrium]: Table 2. Kinetic parameters of lariat debranching reactionsa Molecules Kdb (nM) kobsc (per min) Fraction of productd EBS3 (293) G (wt) 420 ± 60 1.28 ± 0.06 (1.26e) 0.186 ± 0.009 (0.172e) A 100 ± 20 1.84 ± 0.11 (1.93e) 0.166 ± 0.010 (0.159e) U 690 ± 110 1.20 ± 0.10 (1.28e) 0.153 ± 0.010 (0.189e) C 2200 ± 200 1.23 ± 0.06 (1.68e) 0.138 ± 0.005 (0.143e) δ–δ′ (252:195) C:G (wt) 420 ± 60 1.28 ± 0.06 0.186 ± 0.009 U:A 3200 ± 400 1.58 ± 0.10 0.144 ± 0.006 C:Gf (wt) 0.82 ± 0.14 0.169 ± 0.011 0.114 ± 0.007 C:Af 870 ± 60 0.198 ± 0.011 0.120 ± 0.003 a Purified lariat intron samples were used (Scheme 2). Values are for the rE5 5′ exon. b See Materials and methods and Costa and Michel (1999). c Observed rate of lariat debranching (kdeb,I + kbr,I, Scheme 2) at a saturating concentration of 5′ exon. d Final fraction of debranched molecules at a saturating concentration of 5′ exon. e Values in parentheses were obtained by quantitating all intron-containing products in reactions of IE3 lariat intermediate with the rE5 exon (Figure 4A) and extracting rate constants for intron debranching and branching (IE3 molecules had matched EBS3:IBS3 combinations). f Temperature was 25°C instead of 40°C. Exon ligation is nevertheless observed to proceed to completion (Figure 4A) when the concentration of 5′ exon is much higher than that of IE3. Under such conditions, controls showed that rates for each individual step (Figure 4A) are independent of the initial intron concentration (Materials and methods), as should be the case for pseudo-first-order reactions. Figure 4.Reactivity of IE3 lariat intermediate molecules with matched and mismatched base combinations at positions 293 (EBS3) and +1 (IBS3). (A) Example of reaction kinetics. Purified 293A:+1U IE3 molecules (20 nM) were reacted (Materials and methods) with 20 μM 2′dE5 exon (Table I). Experimental data (fractions of the initial molar concentration of IE3 as a function of time) were fitted with the help of the Kintecus software (J.Ianni) to curves generated from the set of linear differential equations corresponding to Schemes 1 and 2. Open circles and the continuous curve (—), IE3; open diamonds and long dashes (– – –), E5IE3; open squares and small dashes (---), lariat intron (I); filled diamonds and dotted curve (…), E5I. (B) Calculated rate constants of exon ligation (klig, Scheme 1) for IE3 molecules with diverse 293:+1 base combinations. Conditions as in (A) with 5 μM rE5 5′ exon (thick horizontal black bars) and 20 μM 2′dE5 (open bars) for all combinations except those including C293, for which 25 μM rE5 and 100 μM 2′dE5 were used (vertical lines connect black and open bars for the same mutant). Reactions with the rE5 exon of IE3 molecules carrying matched EBS3–IBS3 combinations were somewhat too rapid for their rate to be estimated precisely. (C) Relative rate of debranching over branching for IE3 molecules. The exon was 2′dE5. Ratios are expressed as kdeb,IE3/(kbr,IE3 + kdeb,IE3) (see Scheme 1) in order to allow comparison with final fractions of debranched lariat intron in Table II [note that the wild-type lariat intron is debranched more extensively (final fraction ∼0.38) by the 2′dE5 than the rE5 exon]. Download figure Download PowerPoint As shown in Figure 4B, the rate constant (klig) of exon ligation is much decreased by mutations at either position 293 or +1 (splicing of mutated molecules nevertheless remains faithful, as judged from the migration of the ligated exon product in denaturing gels; data not shown). Unfortunately, the reaction of the wild-type IE3 molecule with an all-ribose (rE5, Table I) 5′ exon is too rapid, even at pH 5.8, for its rate to be estimated accurately. However, ligation can be slowed down ∼50-fold by removing the 2′-OH group at exon position −1 (Podar et al., 1998; Dème et al., 1999) and, in this context, single base substitutions at positions 293 and +1 are seen to reduce klig further by 100- to 1000-fold (Figure 4B). Under these conditions, it also becomes apparent that efficient ligation is largely restored by combining those single substitutions so as to generate Watson–Crick combinations other than the wild-type one (as expected for a canonical base pair, the G293:U+1 wobble combination displays somewhat intermediate behavior). These experiments thus demonstrate both the existence of the 293(EBS3):+1(IBS3) pair and its importance for the second step of splicing. In contrast to their impairment of exon ligation, base substitutions at position 293 have only moderate (<5-fold) effects on the Kd for the 5′ exon and essentially no influence on the rate of production and final (equilibrium) fraction of debranched molecules when assayed in the context of the lariat intron [Scheme 2 and Table II; Kd was estimated from the dependence of the final fraction of debranched product on 5′ exon concentration, as explained in Costa and Michel (1999)]. Since Scheme 2 is related to the first step of splicing, the latter thus appears essentially unaffected. Nevertheless, all combinations other than G:C and C:G show somewhat elevated rates of debranching of the IE3 form (compared with branching, Figure 4C), which most probably reflects the fact that the EBS3–IBS3 base pair, which is shown by kethoxal modification to exist in the complex between the lariat intron and ligated exons (Figure 2A), is also part of the ground state of the wild-type IE3 molecule [disruption or weakening of another interaction that is believed to come into existence only after the first step of splicing was reported by Chanfreau and Jacquier (1996) to have the same consequences in the related Sc.a5γ intron from yeast mitochondria]. Statistical evidence for the EBS3–IBS3 and δ–δ′ pairings Only one residue separates G293 from the 3′ branch of helix ID(iv), whose length and sequence tend to be rather well conserved in subgroup IIB introns (Figure 1B in Michel et al., 1989). It is therefore straightforward to align available subgroup IIB sequences locally and check whether the base homologous to nucleotide 293 is generally constrained to pair with the first nucleotide of the 3′ exon. As seen in Table III, this is clearly the case, since a Watson–Crick base pair could form in 53 out of 69 sequences (mutual information 0.593). Moreover, in five of the remaining sequences, a G:U pair could exist. Table 3. Statistical evidence for EBS3–IBS3 and δ–δ′ pairings in group IIB intronsa EBS3 (293 and counterparts) IBS3 A C G U A 1 1 2 18 C 2 0 1 0 G 0 18 0 5 U 16 2 1 2 δ δ′ (195 and counterparts) A C G U A 3 0 0 0 C 2 0 16 0 G 0 8 1 0 U 30 6 1 2 a The same 69 sequences were analyzed as in Figure 1B. Pairing of G293 with the first base of the 3′ exon implies that nucleotide 252 (δ), immediately 5′ of EBS1, lies relatively close to it during the ligation step. We therefore sought a possible partner for C252 among the bases that are part of the internal loop between helices ID(iii) and ID(iv). As seen in Table III, nucleotide 195, immediately 5′ of the 5′ branch of helix ID(iv), tends to co-vary with the base at position 252 (mutual information 0.548), and indeed seems capable of forming a canonical base pair with the latter [54 out of 69 sequences; the lack of A(δ):U(δ′) combinations could reflect the necessity to avoid extending the ID(iv) helix by base pairing between nucleotide 195 and the well-conserved A at position 292]. Disruption of the δ–δ′ pair interferes with exon binding In order to assess the possible existence of a C252(δ)–G195(δ′) base pair in the Pl.LSU/2 intron, we generated molecules with U:A and G:C combinations at these two sites and compared them both with those carrying single substitutions and the wild type. While mutant precursor transcripts were sufficiently reactive to generate workable amounts of excised intron, all purified lariat intron molecules except U252–A195 proved unable to debranch significantly (Scheme 2) when confronted with 5 μM rE5 5′ exon at 40°C. Since closer examination of the kinetic parameters for the debranching reaction of the U:A lariat revealed a marked increase in Kd (Table II), we sought conditions that would allow tighter binding of the 5′ exon. As seen in Figure 5A, not only does the Kd for dissociation of the 5′ exon from wild-type lariat molecules show the expected strong dependence on temperature, but the Van't Hoff plot of log(Kd) versus 1/T appears linear between 25 and 45°C, which suggests that neither the lariat nor the 5′ exon undergo conformational changes liable to interfere with their mutual recognition within the temperature range investigated. The overall decrease in Kd between 40 and 25°C is ∼500-fold and, when assayed at the latter temperature in the presence of 5 μM rE5, all mutated lariats were found to debranch to a significant extent (Figure 5B), in keeping with the possibility that their Kd was now either below or in the same range as the concentration of 5′ exon. That this was the case was verified by determining the Kd for the 252C:195A lariat, which although ∼1000-fold higher than for the wild type, is nevertheless <5 μM at 25°C (Table II). Figure 5.Reactivity of lariat intron molecules with matched and mismatched base combinations at positions 252 (δ) and 195 (δ′). (A) Van't Hoff plot of Kd for dissociation of the rE5 5′ exon from the wild-type lariat (252C:195G). Kd was estimated from the dependence of the final fraction of debranched product on rE5 concentration (Costa and Michel, 1999). (B) Final fraction of debranched molecules at 5 μM rE5 as a function of temperature. Download figure Download PowerPoint In order to characterize the other mutated molecules, we resorted to the simple test of determining the final fraction of debranched lariat as a function of temperature for a constant 5 μM concentration of 5′ exon: as the temperature is raised, this fraction is seen (Figure 5B) to decrease progressively to undetectable levels, with the temperature at mid-transition presumably corresponding to that at which Kd becomes approximately equal to 5 μM. By this criterion, the C:A combination appears less fit to bind the exon than U:G, which fares, in turn, less well than U:A and, finally, C:G. While this order is of course the expected one for a Watson–Crick pair, exchanging G and C results in a molecule that loses the ability to bind the exon at ∼35°C, rather than at ∼47°C for the wild type. However, whatever the reason for the poorer performance of the G:C combination, it nevertheless is seen to offer some significant measure of compensation over C:C and G:G mismatches, which provides a further indication of a canonical interaction between positions 252(δ) and 195(δ′). Is the δ–δ′ pair part of the ground state of the intron? The inability of molecules lacking the δ–δ′ pair to bind their 5′ exon tightly strongly suggests that one function of this interaction is to constrain the structure of the ID3 loop in such a way that the EBS1–IBS1 helix can be stabilized. In order to determine whether formation of the δ–δ′ pair is facilitated by binding of the exon, we probed the wild-type and a C252G mutant lariat with kethoxal in the presence and absence of the unreactive 3′dE5 5′ exon (Figure 2B). In the C252G mutant molecule, the G at position 195 reacts with kethoxal in both the absence and presence of the exon (whose binding was verified to be complete by DMS modification; data not shown). By comparison, G195 is significantly (by a factor of ∼4.5) protected in the wild-type lariat, where it can pair with the C at 252 and, even though its reactivity is reduced further (∼3-fold) upon binding of the exon, this strongly suggests that the δ–δ′ pair exists in the absence of the EBS–IBS pairings (another residue that responds to the presence of the exon is G260, which, although assumed to base pair with U250, is not fully protected unless both the δ–δ′ and IBS1–EBS1 interactions are present). Finally, we did not note any difference in the extents of protection from kethoxal modification afforded to G195 by the 5′ exon and ligated exons (data not shown). Therefore, the EBS3–IBS3 and δ–δ′ base pairs must co-exist in the ground state of the complex between the intron lariat and its exons. Three-dimensional architecture of domains ID and V Subdomain ID has a central role in organizing the active center of group II introns because it includes not only the three exon-binding sites [EBS1, EBS2 and (this work) EBS3], but also two important receptor sites (Figure 3A) for the small and well-conserved domain V, which is regarded as being involved in catalysis. We now detail how a three-dimensional model of the complex formed by the ligated exons, subdomain ID and domain V can be built by combining all available data. Besides confirming the widespread occurrence of the δ–δ′ and EBS3–IBS3 interactions, comparative analysis of available subgroup IIB sequences reveals two other major constraints on the architecture of subdomain ID. A number of subgroup IIB introns lack the EBS2–IBS2 pairing and, in most of these molecules, stems ID(iv) and ID3(i) are fused together (see Figure 3B). This shows that these two helices, which are almost always contiguous when distinct, actually stack end-to-end. A second pair of helices, which are most likely to be coaxial, are ID(ii) and ID(iii). These two stems are part of a three-way junction motif, H(elix)1-A–H(elix)2-GAA–H(elix)3-, which recurs at different locations in self-splicing introns. As shown in Figure 3C, this motif may be replaced by a single continuous helix, equivalent to H1 + H3, and, since the substitution occurs in closely related molecules, it is quite unlikely to change the relative geometry of the sections distal to H1 and H3 (in the example provided, the loops on either side of H1–H3 are well-conserved, important components of domain I). The ID(ii)–ID(ii)a–ID(iii) junction is also of interest as a binding site for domain V. Not only does chemical modification of the adenines of the internal loop interfere with binding of domain V by domain I (Jestin et al., 1997), but these three bases are footprint