The guanosine binding mechanism of the Tetrahymena group I intron.

The Tetrahymena group I intron catalyzes self-splicing through two consecutive transesterification reactions, using a single guanosine-binding site (GBS). In this study, we constructed a model RNA that contains the GBS and a conserved guanosine nucleotide at the 3'-terminus of the intron (omegaG). We determined by NMR the solution structure of this model RNA, and revealed the guanosine binding mechanism of the group I intron. The G22 residue, corresponding to omegaG, participates in a base triple, G22 xx G3 x C12, hydrogen-bonding to the major groove edge of the Watson-Crick G3 x C12 pair. The G22 residue also interacts with A2, which is semi-conserved in all sequenced group I introns.     

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.     

Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site.

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3'-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5'-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3'-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.     

The consensus sequence for self-catalyzed site-specific G residue depurination in DNA

The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme

The 3' splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L-21 Scal ribozyme from Tetrahymena by adding residues to the 5' end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG <--> CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value approximately 10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react approximately 10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5' extensions. Shortening S to include only one residue 3' of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3' portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5' extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.     

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.     

Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures

Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A(4324) in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) into a DNA stem-loop reduced k(cat) by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2'-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k(cat) 20-fold to 1660 min(-1), a value similar to RTA's k(cat) on intact ribosomes.     

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.     

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.     

The methylation of one specific guanosine in a pre-tRNA prevents cleavage by RNase P and by the catalytic M1 RNA.

Several modified nucleosides were introduced during in vitro RNA synthesis into a pre-tRNA(Ser). The pre-tRNAs were used as substrates for RNase P enzymes. No effects were observed with biotin-8-ATP or [alpha-S]-GPT, whereas with m7GTP, the cleavage reaction was completely inhibited. Analysis of pre-tRNAs which contained m7G at various positions has revealed a single base at the 5'-end of the acceptor stem where this modification absolutely prevents cleavage by catalytic M1 RNA, eukaryotic and prokaryotic RNase P holoenzymes. These results suggest that a critical contact must be made between pre-tRNA substrate and enzyme/ribozyme or that the approach of the potential cleaving agent (a positive magnesium ion) is made impossible by the positive charge at N-7 of the guanosine. In addition, we have shown that a pre-tRNA containing only m7G's can still form a complex with M1 RNA in a gel retardation assay.     

Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease

RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.     

The stem-loop binding protein forms a highly stable and specific complex with the 3' stem-loop of histone mRNAs

Replication-dependent histone mRNAs end in a highly conserved 26-nt stem-loop structure. The stem-loop binding protein (SLBP), an evolutionarily conserved protein with no known homologs, interacts with the stem-loop in both the nucleus and cytoplasm and mediates nuclear-cytoplasmic transport as well as 3'-end processing of the pre-mRNA by the U7 snRNP. Here, we examined the affinity and specificity of the SLBP-RNA interaction. Nitrocellulose filter-binding experiments showed that the apparent equilibrium dissociation constant (Kd) between purified SLBP and the stem-loop RNA is 1.5 nM. Binding studies with a series of stem-loop variants demonstrated that conserved residues in the stem and loop, as well as the 5' and 3' flanking regions, are required for efficient protein recognition. Deletion analysis showed that 3 nt 5' of the stem and 1 nt 3' of the stem contribute to the binding energy. These data reveal that the high affinity complex between SLBP and the RNA involves sequence-specific contacts to the loop and the top of the stem, as well the base of the stem and its immediate flanking sequences. Together, these results suggest a novel mode of protein-RNA recognition that forms the core of a ribonucleoprotein complex central to the regulation of histone gene expression.     

NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.     

Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids

A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.     

Crystal structure of a group I intron splicing intermediate

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.