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Gutell 072.jmb.2000.301.0265


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Gutell 072.jmb.2000.301.0265

  1. 1. Function of Tyrosyl-tRNA Synthetase in SplicingGroup I Introns: An Induced-fit Model for Binding tothe P4-P6 Domain Based on Analysis of Mutations atthe Junction of the P4-P6 Stacked HelicesXin Chen, Robin R. Gutell and Alan M. Lambowitz*Institute for Cellular andMolecular Biology, Departmentof Chemistry and Biochemistryand Sections of MolecularGenetics and Microbiology andIntegrative Biology, School ofBiological Sciences, Universityof Texas at Austin, AustinTX 78712, USAWe used an Escherichia coli genetic assay based on the phage T4 td intronto test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNAsynthetase (CYT-18 protein) to suppress mutations that cause structuraldefects around its binding site in the P4-P6 domain of the group I introncatalytic core. We analyzed all possible combinations of nucleotides ateither P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6stacked helices, and looked for synergistic effects in double mutants.Most mutations at either position inhibit self-splicing, but can be sup-pressed by CYT-18. CYT-18 can compensate ef®ciently for mutations thatdisrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotidesubstitutions at either position that are predicted to be suboptimal forbase stacking, based on the analysis of DNA four-way junctions. How-ever, CYT-18 has dif®culty suppressing combinations of mutations atP4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and basestacking. Thermal denaturation and Fe(II)-EDTA analysis showed thatmutations at the junction of the P4-P6 stacked helices lead to grosslyimpaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressible mutants bind the protein with Kd values up to 79-foldhigher than that for the wild-type intron, but in all cases tested, the koffvalue for the complex remains within twofold of the wild-type value,suggesting that the binding site can be formed properly and that theincreased Kd value re¯ects primarily an increased kon value for the bind-ing of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substi-tutions grossly disrupt the catalytically-active group I intron tertiarystructure, and that CYT-18 binding induces the formation of the correctstructure in this region, leading to folding of the group I intron catalyticcore.# 2000 Academic PressKeywords: aminoacyl-tRNA synthetase; group I intron; ribozyme;RNA splicing; RNA structure*Corresponding authorIntroductionGroup I introns fold into a conserved tertiarystructure that is required for catalytic activity(Michel & Westhof, 1990; Cech & Golden, 1999).Although some group I introns are self-splicingin vitro, most, if not all, require proteins to facilitateRNA folding in vivo (reviewed by Lambowitz &Perlman, 1990; Lambowitz et al., 1999). These pro-teins either bind speci®cally to the intron RNAs(Weeks & Cech, 1995; Shaw & Lewin, 1995;E-mail address of the corresponding author:lambowitz@mail.utexas.eduAbbreviations used: DMS, dimethyl sulfate; Kan,kanamycin; LSU, large subunit ribosomal RNA; MM,minimal medium; MMT, minimal medium plusthymine; mt, mitochondria; ORF, open reading frame;phenol-CIA, phenol/chloroform/isoamyl alcohol(25:24:1); TS, thymidylate synthase; TTM, minimalmedium plus thymine and trimethoprim; TyrRS,tyrosyl-tRNA synthetase.doi:10.1006/jmbi.2000.3963 available online at on J. Mol. Biol. (2000) 301, 265±2830022-2836/00/020265±19 $35.00/0 # 2000 Academic Press
  2. 2. Saldanha et al., 1996; Ho et al., 1997) or interactnon-speci®cally as RNA chaperones (Coetzee et al.,1994; Zhang et al., 1995). The Neurospora crassamitochondrial tyrosyl-tRNA synthetase (mtTyrRS), or CYT-18 protein, functions in splicing bybinding speci®cally to the group I intron catalyticcore (Guo & Lambowitz, 1992; Caprara et al.,1996a,b). The CYT-18 protein normally functions insplicing the mt large rRNA (mt LSU) intron andother group I introns in Neurospora mitochondria(Mannella et al., 1979; Akins & Lambowitz, 1987;Wallweber et al., 1997). However, it can also bindto and splice diverse group I introns from otherorganisms, including introns belonging to differentstructural subgroups, so long as its binding site inthe catalytic core is not obscured by peripheralRNA structures (Guo & Lambowitz, 1992; Mohret al., 1992, 1994). As discussed below, the splicingfunction of CYT-18 may re¯ect its ability to recog-nize conserved tRNA-like structural features of thegroup I intron catalytic core (Caprara et al., 1996b).The group I intron catalytic core consists of twoextended helical domains: the P4-P6 domainformed by the coaxial stacking of the secondarystructure elements P5, P4, P6 and P6a, and the P3-P9 domain formed by P8, P3, P7 and P9 (Figure 1;Michel & Westhof, 1990; Golden et al., 1998). Thejuxtaposition of the two domains forms the intronsactive site, which contains the binding sites for the5Hand 3Hsplice sites (helices P1 and P10, respect-ively) and the guanosine cofactor (P7). The orien-tation of the two helical domains is maintained bythe base-triple interactions between the P4-P6domain and the adjoining single-stranded junctionregions J3/4 and J6/7, as well as long-range ter-tiary interactions, such as the base triple betweenP4 bp-5 and J8/7-5 and the tetraloop/minorgroove interaction between L9 and P5 (Michel &Westhof, 1990; Tanner & Cech, 1997). RNA foldingstudies with the Tetrahymena LSU intron indicatethat the P4-P6 domain folds ®rst to form a scaffoldfor the assembly of the P3-P9 domain (Murphy &Cech, 1993; Laggerbauer et al., 1994; Zarrinkar &Williamson, 1994; Scalvi et al., 1998).The N. crassa mt LSU and ND1 introns, whichare dependent on CYT-18 for splicing both in vitroand in vivo, are not detectably self-splicing(Mannella et al., 1979; Garriga & Lambowitz, 1986;Wallweber et al., 1997). RNA structure mappingshowed that these introns could form most of theshort-range helices of the conserved group I intronsecondary structure, but otherwise remain largelyunfolded in the absence of CYT-18. CYT-18restores splicing by binding to the unfolded intronRNAs and promoting formation of the catalyti-cally-active group I intron tertiary structure(Caprara et al., 1996a,b). RNA footprinting exper-iments with the N. crassa mt LSU and ND1 intronsshowed that CYT-18 protects the phosphodiesterbackbone on the surface of the catalytic core oppo-site the active-site cleft. Most of the putative CYT-18 protection sites were in the P4-P6 domain, clus-tered around the junction of the P4-P6 stackedhelix, but additional sites were found in P3, P8 andP9 in both introns and in P7.1/P7.1a and L9 in themt LSU intron (Caprara et al., 1996a). Consistentwith these results, a small RNA containing onlythe P4-P6 domain of the mt LSU intron could bindCYT-18 independently (Kd ˆ 130 pM at 22 C), butadditional sequences from the P3-P9 domain wererequired for maximal binding (Kd ˆ 22 pM; Guo Lambowitz, 1992; Saldanha et al., 1996). By con-trast, the isolated P3-P9 domain could not bindCYT-18 independently, presumably because it doesnot fold correctly in the absence of the P4-P6domain (Saldanha et al., 1996). Together, these stu-dies suggested a model in which CYT-18 binds®rst to the P4-P6 domain to form a scaffold for theassembly of P3-P9 and then makes additional con-tacts with the P3-P9 domain to stabilize the twohelical domains in the correct relative orientationto form the introns active site (Caprara et al.,1996a,b).This model for CYT-18 function is supportedfurther by studies with the Tetrahymena LSUintron. The Tetrahymena LSU intron contains alarge peripheral RNA structure, P5abc, which ordi-narily blocks the CYT-18 binding site in the P4-P6domain. The deletion of the P5abc structure resultsin loss of self-splicing at low Mg2‡concentrations,but splicing can be restored by adding either theCYT-18 protein or P5abc RNA in trans (van derHorst et al., 1991; Mohr et al., 1994). CYT-18 andP5abc bind to overlapping sites around the junc-tion of the P4-P6 stacked helices and likely playsimilar roles in stabilizing the structure of the P4-P6 domain, exemplifying how a protein mightassume a role played by an RNA in the course ofevolution (Mohr et al., 1994).Remarkably, comparison of the CYT-18 bindingsites in the N. crassa mt LSU and ND1 introns withthat in N. crassa mt tRNATyrby RNA footprintingin conjunction with graphic modeling revealed anextended three-dimensional overlap between thetRNA and the group I intron catalytic core. In thisoverlap, the P4-P6 domain corresponds to theanticodon/D-arm stacked helices of the tRNA, P7to the long variable arm, and P9 to the acceptorstem (Caprara et al., 1996b). These ®ndings supportthe hypothesis that CYT-18 adapted to function insplicing by recognizing conserved tRNA-like struc-tural features of group I introns, and raise thepossibility of an evolutionary relationship betweengroup I introns and tRNAs.The interaction of CYT-18 with the P4-P6domain, the cognate of the tRNAs anticodon/D-arm stacked helices, is critical to its role in splicing.To investigate this interaction in detail, we tookadvantage of the ability of CYT-18 to bind speci®-cally to the isolated P4-P6 domain of the N. crassamt LSU intron (Guo Lambowitz, 1992; Saldanhaet al., 1996). In vitro selection experiments to ident-ify features required for CYT-18 binding identi®edten invariant residues around the junction of theP4-P6 stacked helices, including the four residuescomprising P4 bp-1 and P6 bp-1 at the stacked266 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  3. 3. helical junction, as well as P4 bp-3, P6 bp-2, J3/4-1and J3/4-2 (Saldanha et al., 1996). RNA footprint-ing and modi®cation-interference experimentsshowed that the interaction of CYT-18 with the iso-lated domain is similar to that in the intact intron,with phosphate backbone protections on both sidesof the junction of the P4-P6 stacked helix, but fewif any base-speci®c contacts in this region (M.G.Caprara A.M.L., unpublished results). Finally, itwas shown that CYT-18 could not bind to a per-muted version of the P4-P6 domain, in which P4-P6 is a continuous rather than a stacked helix,suggesting that the con®guration recognized byCYT-18 is not perfectly coaxial (Saldanha et al.,1996).Previously, we developed an E. coli genetic assaythat permits us to identify rapidly structuraldefects that can or cannot be suppressed by CYT-18. This assay is based on the ability of CYT-18 torestore the splicing of structurally-defectivemutants of a plasmid-borne phage T4 td intron(Mohr et al., 1992). Splicing of the intron leads tothe synthesis of thymidylate synthase, whichenables thyAÀcells to grow on minimal mediumand confers sensitivity to trimethoprim on mediumcontaining thymine. By using this assay, weshowed that CYT-18 could suppress splicing-defec-tive td introns containing mutations in differentregions of the catalytic core, including J3/4, P4, P6,J6/7, P7, P8 and P9 (Mohr et al., 1992; Myers et al.,1996). Here, we further tested our model of CYT-18 function by using this bacterial genetic assay toinvestigate systematically the effect of nucleotidesubstitutions at the junction of the P4-P6 stackedhelices. Our results indicate that the P4/P6 junctionis a linchpin region, where even small nucleotidesubstitutions grossly disrupt formation of the cata-lytically-active group I intron tertiary structure,and that CYT-18 binding induces the formation ofthe correct structure in this region, leading to fold-ing of the group I intron catalytic core.ResultsAnalysis of mutations at P4 bp-1The experimental strategy was to introducemutations at P4 bp-1 and/or P6 bp-1 of the tdFigure 1. Secondary-structuremodel of the phage T4 td intron.The td intron (Genebank accessionnumber M12742) belongs to struc-tural subgroup IA. TheFigure shows the predicted second-ary structure of the 265 nt ÁORFderivative of the td intron(pTZtd1304) used in this study. Thestructure is drawn in the format ofCech et al. (1994) and numberedaccording to Belfort et al. (1987).Nucleotide residues in the intronand exons are indicated in upperand lowercase letters, respectively.The 5Hand 3Hsplice sites (5HSS and3HSS) are indicated by arrows. Ter-tiary interactions are indicated bythin connecting lines. ÁORF indi-cates the deleted region containingthe intron ORF. Nucleotide resi-dues changed to create restrictionsites are circled. The box indicatesnucleotide residues in P4 bp-1 andP6 bp-1 that were mutagenizedhere.Tyrosyl-tRNA Synthetase in Group I Intron Splicing 267
  4. 4. intron, and then use the E. coli genetic assay torapidly identify those with splicing defects thatcould or could not be suppressed by CYT-18.pTZtd plasmids carrying a phage T4 td gene withmutations at the P4-P6 junction region were trans-formed into an E. coli thyAÀstrain in the presenceor absence of pA550, which expresses wild-typeCYT-18 protein. The transformants were then pla-ted on minimal medium (MM), minimal mediumplus thymine (MMT), and minimal medium plusthymine and trimethoprim (TTM) (Belfort et al.,1987; Mohr et al., 1992). Cells harboring splicing-defective td introns lack functional TS and areunable to grow on MM, but grow on MMT andare insensitive to trimethoprim. Restoration of spli-cing by CYT-18 results in the synthesis of TS,which enables the cells to grow on MM and con-fers sensitivity to trimethoprim. Previous studiesshowed a good correlation between the growthphenotype in the plating assay and the amount ofspliced td mRNA, with 1 % splicing giving a tdÀphenotype and 38 % splicing giving a fully wild-type phenotype (Mohr et al., 1992; Brion et al.,1999b).Results of td plating assays for all 16 possiblenucleotide combinations at P4 bp-1 are summar-ized in Table 1A, and representative plating assaysare shown in Figure 2. The wild-type intron, whichcontains an AU base-pair at P4 bp-1, gave atd‡phenotype in the presence or absence ofCYT-18, indicating that it could splice ef®cientlyunder both conditions. Most of the P4 bp-1 mutantintrons gave tdÀphenotypes in the absence ofCYT-18, indicating that they are unable to self-splice ef®ciently, but gave td‡phenotypes indica-tive of restored splicing in the presence of CYT-18.Two mutants, Aa and ua, showed weak splicing inthe absence of CYT-18, and two other mutants, Agand gg, showed no detectable splicing in the pre-sence or absence of CYT-18. (Note: wild-typenucleotide residues are indicated in uppercaseletters and mutant nucleotide residues in lowercaseletters.) The ®nding that self-splicing was inhibitedby nearly all nucleotide substitutions, includingthose that retain the ability to form a Watson-Crickor a wobble base-pair (cg, gc, gU, ug), indicates arequirement for speci®c nucleotide residues atP4 bp-1. Indeed, this base-pair is highly con-strained, corresponding to AU or UA in 98 % ofall naturally occurring group I introns (see Discus-sion). Since nearly all of the P4 bp-1 mutants canbe ef®ciently suppressed by CYT-18, we infer thatthe structural defect resulting from the nucleotidesubstitutions can be compensated for by the pro-tein, and that CYT-18 does not make an indispen-sable base-speci®c contact with P4 bp-1.The two non-splicing mutants, which could notbe rescued by CYT-18, are the purine-purine mis-matches Ag and gg. However, the other two pur-ine-purine mismatches, Aa and ga, could berescued. The difference could re¯ect that depend-ing on sequence context purine-purine mismatchescan potentially form either an imino hydrogen-bonded pair or a sheared base-pair (Wu Turner,1993; Chou et al., 1997). The latter is not readilyaccommodated in a standard A-form helix and cancause structural disruption by changing the widthsof the major and minor grooves (Wu Turner,1993; Gautheret et al., 1994). In addition, the non-splicing mutants gg and Ag can potentially forman alternative, presumably non-productive second-ary structure, which is not predicted for the CYT-18 suppressible mutants Aa and ga (Figure 3).Chemical structure mapping with DMS indicatedthat this alternative secondary structure does infact form in the mutants (see Figure 3).Analysis of mutations at P6 bp-1An analogous set of mutants was constructed forP6 bp-1. Unlike P4 bp-1, this base-pair is involvedin a known tertiary interaction, a minor-groovenucleotide triple with J3/4-3, which imposesadditional sequence constraints (Figures 1 andTable 1. In vivo splicing phenotypes of td intronmutantsSplicing phenotypeMutants ÀCYT-18 ‡CYT-18A. P4 bp-1AU (WT) ‡ ‡ ‡ ‡ ‡ ‡Aa ‡ ‡‡Ac À ‡ ‡ ‡Ag À Àca À ‡ ‡ ‡cc À ‡ ‡ ‡cg À ‡ ‡ ‡cU À ‡ ‡ ‡ga À ‡‡gc À ‡ ‡ ‡gg À ÀgU À ‡ ‡ ‡ua ‡ ‡ ‡ ‡uc À ‡ ‡ ‡ug À ‡‡uU À ‡ ‡ ‡B. P6 bp-1GC (WT) ‡ ‡ ‡ ‡ ‡ ‡aa À ÀaC À ‡‡ag À ‡au ‡ ‡‡ca À ‡ ‡ ‡cC À ‡cg ‡ ‡ ‡ ‡cu À ‡ ‡ ‡Ga À ‡‡Gg À ‡‡Gu À ‡‡ua À ‡‡uC À ‡‡ug À ‡‡uu À ‡This summarizes in vivo splicing phenotypes of the wild-type(WT) and mutant td introns having nucleotide substitutions ateither (A) P4 bp-1 or (b) P6 bp-1, in the presence (‡) or absence(À) of the CYT-18 protein, based on plating assays. Wild-typenucleotide residues are indicated in uppercase letters, andmutant nucleotide residues are indicated in lowercase letters.Splicing phenotypes are as de®ned in Materials and Methods.268 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  5. 5. 4(a)). In a previous mutational analysis of P6 bp-1using the full-length td intron, Ehrenman et al.(1989) found that the mutants cC and Gg could notself-splice in vivo and that the compensatorymutant cg only weakly restored splicing, implyingeither additional structural constraints or a dualfunction of this base-pair.The results of the td plating assay for the morecomplete set of P6 bp-1 mutants analyzed here aresummarized in Table 1B. The wild-type intron,which has a GC base-pair at this position, splicedef®ciently in the presence or absence of CYT-18.Again, most of the mutants failed to splice in theabsence of CYT-18, but could be suppressed by theprotein. The only two mutants that showed weaksplicing in the absence of CYT-18 were cg, asreported by Ehrenman et al. (1989), and au. Both ofthese mutants retain a canonical base-pair atP6 bp-1, as well as nucleotide triple combinationsthat are found in naturally occurring group Iintrons (A-U:A and C-G:A; Figure 4(b,c), Table 2).The mutant ua, which also retains a Watson-Crickbase-pair at P6 bp-1, could not self-splice and wasnot fully suppressed by CYT-18. Examination ofpotential base triples using the ISOPAIR program(Gautheret Gutell, 1997) showed that U-A:Acould form an alternative, more stable base triplethat would disrupt the normal phosphodiesterbackbone geometry, possibly accounting for theimpaired self-splicing of the mutant (Figure 4(d)).This alternative base-triple con®guration is foundnaturally in yeast tRNAPhe(U12:A23:A9) (Quigley Rich, 1976). If this interpretation is correct, theU-A:A combination may be deleterious in group Iintrons, and indeed it has been reported in onlytwo subgroup IB mutants of unknown self-splicingcapability (Table 2). Mutants Gu and ug, whichintroduce a wobble base-pair at Pb bp-1, did notself-splice and were only moderately suppressedby CYT-18. Since CYT-18 can suppress nearly allsubstitutions at P6 bp-1, we infer that it can com-pensate generally for impaired base-pairing andbase-stacking interactions, as well as for the loss ofthe normal base-triple interaction with J3/4-3, con-sistent with previous analysis of J3/4 mutations(Myers et al., 1996). In addition, as for P4 bp-1, wecan infer that CYT-18 makes no indispensablebase-speci®c contacts with P6 bp-1.CYT-18 had particular dif®culty suppressingP6 bp-1 mutants aa, ag, cC, and uu. The dif®cultyin suppressing purine-purine mismatches is remi-niscent of the situation at P4 bp-1. However, themost deleterious mutation at P6 bp-1 was aa,rather than ag or Gg, which were the most deleter-ious at P4 bp-1. Again, a possible explanation isthat aa in this sequence context forms a shearedFigure 2. Plating assay for spli-cing of P4 bp-1 and P6 bp-1mutants in the presence or absenceof the CYT-18 protein. E. coli 1904(C600 thyA::Kanr) containing wild-type or mutant pTZtd plasmidsplus pA550 (‡CYT18) or the vectorpACYC184 (ÀCYT18) were platedon MMT, MM and TTM containingkanamycin, chloramphenicol andampicillin and grown overnight at37 C. The grids at the top showthe pattern of P4 bp-1 (left) andP6 bp-1 (right) mutants inoculatedon each plate. The negative control,pTZtdÁP4-7, contains an intronwith an internal deletion, whichdoes not splice in the presence orabsence of CYT-18 (Mohr et al.,1992).Tyrosyl-tRNA Synthetase in Group I Intron Splicing 269
  6. 6. purine-purine pair, thereby causing greater distor-tion of the major and minor grooves. Unfavorablebase-stacking interactions may also contribute,since aa, along with the pyrimidine-pyrimidinemismatches uu, cu, and uc, have been found tocause the greatest disruption of base stacking instudies with DNA four-way junctions (Duckett Lilley, 1991). In extrapolating these results to RNA,the tacit assumption is that stacking preferencesare determined largely by base interactions (seeDuckett et al., 1995). However, cu and uC, whichare also expected to be unfavorable for base stack-ing, were more readily suppressed than cC and uuat P6 bp-1. The difference here could re¯ect that cuand uC have greater propensity for base-pairingthan uu or cC in certain sequence contexts (cf.Aboul-ela et al., 1985; Werntges et al., 1986). Thisexplanation is compatible with the conclusionbelow that both base-pairing and base stackingcontribute to the formation of the correct geometryat the junction of the P4-P6 stacked helix.Analysis of double mutants at P4 bp-1 andP6 bp-1In order to assess the possible interactionbetween P4 bp-1 and P6 bp-1, we analyzed 26mutants selected randomly from a pool havingmutations at all four positions. The results of tdplating assays are summarized in Table 3. None ofthe mutants was self-splicing. A total of 13 mutantswere CYT-18 dependent, and the remaining 13 didnot splice in the presence or absence of CYT-18. All13 CYT-18-dependent mutants retain at least oneWatson-Crick or wobble base-pair at either P4 bp-1or P6 bp-1, and indeed six of these retain the wild-type base-pair at one of these positions. Only twoof the CYT-18-suppressible mutants, gc/ua andua/ug, have substitutions at all four positions, andboth retain the ability to form Watson-Crick orwobble base-pairs at both P4 bp-1 and P6 bp-1.Interestingly, two of the CYT-18-dependentmutants, gc/ua and cU/au, have combinations ofnucleotides that spliced more ef®ciently than theFigure 3. Alternative secondary structure of P4 bp-1Ag and gg mutants. The predicted secondary structureof the P4-P6 domain of the wild-type td intron is shownto the left, and the alternate structure predicted for theP4 bp-1 Ag and gg mutants is shown to the right.Nucleotides at positions 48 and 77, which compriseP4 bp-1 in the wild-type intron are boxed. Open circlesindicate nucleotide positions modi®ed by DMS inP4 bp-1 Ag and gg mutants, but protected in wild-typeand other P4 bp-1 purine-purine mutants (5®vefolddifference in band intensity). Filled circles indicate pos-itions protected in the P4 bp-1 Ag and gg mutants, butstrongly modi®ed in wild-type and other P4 bp-1 pur-ine-purine mutants (5tenfold difference in band inten-sity). The ®lled triangles indicate positions modi®ed inboth the wild-type and mutant introns.Figure 4. Diagram of potential base-triple interactionsbetween P6 bp-1 and J3/4-3 in wild-type and mutant tdintrons. (a) Wild-type, G-C:A, (b) mutant c-g:A, (c)mutant a-u:A, (d) mutant u-a:A. Base-triple con®gur-ations were predicted by the ISOPAIR program(Gautheret Gutell, 1997). The Figure shows that theU-A:A combination could form an alternate triple,which has a greater number of hydrogen bonds, butwould distort the phosphodiester backbone geometry inthis region. The equilibrium arrows with a questionmark represent the hypothesis that this alternate con-®guration might be favored in the td intron, accountingfor the impaired self-splicing of the P6 bp-1 ua mutant.Numbers indicate nucleotide positions in the td intron.Broken lines indicate hydrogen bonds. Filled pentagonsindicate the position and orientation of ribose sugars.270 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  7. 7. corresponding P6 bp-1 single mutants ua and au(cf. Table 1).All 13 non-splicing mutants had P4 bp-1 andP6 bp-1 substitutions that were individually rescu-able by CYT-18. Thus, their non-splicing pheno-type must result from cumulative effects ordisfavored nucleotide combinations at the two pos-itions. Notably, all of the non-splicing mutants con-tain at least one of the combinations UU, CU, UC,or AA that are expected to interfere most stronglywith base-stacking interactions, based on analysisof DNA four-way junctions (Duckett Lilley,1991). Further, except for Aa/Gg, which containspurine-purine mismatches at both positions, all ofthese non-splicing double mutants contain at leastone pyrimidine-pyrimidine mismatch at eitherP4 bp-1 or P6 bp-1.In addition to the possible effects on base stack-ing, seven of the double mutants that could not berescued by CYT-18 lack a Watson-Crick or wobblebase-pair at either position. Of the remaining sixmutants, four have a Watson-Crick or wobble pairat P4 bp-1 coupled with a pyrimidine-pyrimidinemismatch at P6 bp-1, and two have a wobble pairat P6 bp-1 coupled with a pyrimidine-pyrimidinemismatch at P4 bp-1. Together, these ®ndingssuggest that, in addition to the nucleotide triplewith J3/4, both base-pairing and base-stackingcontribute to establishing the functional geometryat the junction of the P4 and P6 helices, and thatdisruption of base-pairing at P4 bp-1 and/orP6 bp-1 makes base-stacking defects more dif®cultto rescue by CYT-18. The intron appears to be par-ticularly sensitive to disruption of the base-pair atP6 bp-1, since 11 of the 13 non-splicing mutantslack a Watson-Crick base-pair at this position, andthe remaining two have only a wobble pair. Thegreater sensitivity to the disruption of P6 bp-1likely re¯ects that P6 in the td intron is only twobase-pairs long.In vitro splicingTo reinforce the conclusions from the in vivo spli-cing assays, several mutants were tested for theirability to splice in vitro in the presence or absenceof CYT-18 protein in reaction medium containing3 mM Mg2‡, which is near the physiological con-Table 2. Base-pair frequencies at P6 bp-1 in naturally occurring group I introns with an A at J3/4-3P6 bp-1cSubgroupaA at J3/4-3bGC GU AU UA AC CG UGA 20/116 12 7 1B 102/117 87 10 2 2 1C1-2 0/318C3 1/321 1D 14/17 2 11 1E 11/59 6 5aIntron classi®cation based on Michel Westhof (1990) and Suh et al. (1999).bProportion of group I introns in indicated subgroup with an A at J3/4-3.cNumber of group I introns in subgroup with indicated base-pair at P6 bp-1.Table 3. In vivo splicing phenotype of P4-P6 mutantsCYT-18 suppressible Non-splicingMutants Splicing phenotype MutantsP4 P6 ÀCYT-18 ‡CYT-18 P4 P6AU/GC (WT) ‡ ‡ ‡ ‡ ‡ ‡ cg/uCAU/Gu À ‡‡ ug/uuAU/aC À ‡‡ gU/uCAU/uC À ‡‡ gU/uuAU/Gg À ‡‡ uU/GugU/GC À ‡ ‡ ‡ Aa/GgcU/GC À ‡ ‡ ‡ Ac/uCcU/ua À ‡‡ cU/cCuc/Gu À ‡‡ uU/caus/cC À ‡ uc/cucg/Gg À ‡‡ ga/uugc/ua À ‡ ‡ ‡ uc/Ggus/ug À ‡ cU/ugcU/au À ‡ ‡ ‡This summarizes in vivo splicing phenotypes of P4 bp-1/P6 bp-1 double mutants that were suppressed byCYT-18 (left) or were non-splicing in the presence or absence of CYT-18 (right). Uppercase letters indicate wild-type nucleotide residues, and lowercase letters indicate mutant nucleotide residues. Splicing phenotypes are asde®ned in Materials and Methods.Tyrosyl-tRNA Synthetase in Group I Intron Splicing 271
  8. 8. centration (Lusk et al., 1968). As expected, thewild-type td intron spliced ef®ciently under theseconditions in the absence or presence of CYT-18(kobs ˆ 0.18 and 0.19 minÀ1, respectively; 90 %spliced). In agreement with the results of the plat-ing assays, the CYT-18-suppressible P4 bp-1mutants cg, gc, gU, ug, and Ac and the P6 bp-1mutants Gg, cu, and uC showed little, if any,splicing in the absence of CYT-18, but splicedrelatively rapidly in the presence of CYT-18(kobs ˆ 0.053-0.085 minÀ1for the P4 bp-1 mutants,and 0.041-0.056 minÀ1for the P6 bp-1 mutants).These rates of splicing in the presence of CYT-18are in agreement with those previously reportedfor CYT-18-suppressible mutants in other regionsof the td intron (Myers et al., 1996). Notably, a sub-stantial proportion of mutant RNAs (42-61 % forthe P4 bp-1 mutants and 60-65 % for the P6 bp-1mutants) failed to splice even in the presence ofhigh concentrations of GTP (5 mM) and a tenfoldmolar excess of CYT-18, indicating a greater pro-pensity for these mutants to fold into an inactiveRNA conformation. As expected, the non-splicingdouble mutants Ac/uC, cU/ug, uU/Gu, cg/uC,and gU/uC all failed to splice in the presence orabsence of CYT-18. Together, these ®ndings showa very good correlation between the in vitro andin vivo splicing phenotypes for the P4 bp-1 andP6 bp-1 mutants, as found previously for mutantsin other regions of the td intron (Myers et al., 1996).Thermal denaturation analysisIn general, the P4 and P6 mutants could inhibitsplicing either by affecting the global folding of theintron RNA or by creating local structural defectsat the introns active site. To investigate these pos-sibilities, we analyzed the thermal stability of themutant introns by UV spectroscopy. In thisapproach, RNA denaturation is followed by theincrease in absorbance at 260 nm as the tempera-ture is increased slowly from 25 C to 80 C. GroupI introns show an initial structural transition due todisruption of tertiary structure and a later tran-sition due to disruption of secondary structure(Banerjee et al., 1993; Jaeger et al., 1993; Brion et al.,1999a,b). This approach was used by Brion et al.(1999b) to analyze the effect of mutations affectingvarious tertiary interactions on melting pro®les ofthe catalytic core of the td intron, synthesized as anin vitro transcript containing A37 to A1011. TheRNA used here is identical to that used by Brionet al. (1999b), except for the single nucleotidechange U976G, which introduces an EcoRI site forcloning of the P4-P6 mutants (see Materials andMethods and Figure 1).Figure 5 shows the UV-melting pro®les for thecore regions of the wild-type and selected P4 bp-1and P6 bp-1 mutant introns in reaction mediumcontaining 3 mM Mg2‡, the same Mg2‡concen-tration used in the in vitro splicing assays (seeabove). The plots show the ®rst derivative of theabsorbance as a function of temperature, and Tmand ÁTm values derived from the plots are sum-marized in Table 4. The wild-type intron shows aninitial structural transition at 49.6 C due to disrup-tion of tertiary structure and a second transition at67.3 C due to disruption of secondary structure.The Tm for the tertiary structure transition of thewild-type intron is somewhat lower than thatmeasured by Brion et al. (1999a) (52.4 C at 3 mMMg2‡, 56 C at 5 mM Mg2‡), possibly re¯ectingsubtle differences in conditions, or the singlenucleotide difference in the construct used here. Inour experiments, the in-sample digital temperaturesensor was calibrated independently to obtainaccurate temperature readings.Compared to the wild-type intron, the amplitudeof the tertiary-structure transition for all of themutants was decreased, and the transitionsoccurred over a wider temperature range,suggesting decreased cooperativity. Surprisingly,however, in most cases, the Tm for the residual ter-tiary structure was similar to or only slightly lower(3 C) than that for the wild-type intron. Four ofthe P4 bp-1 mutants behaved differently, with thethermal denaturation pro®les showing two rela-tively low temperature transitions (Table 4). Theseinclude the two non-splicing mutants Ag and gg,which were found by chemical structure mappingto form an alternative secondary structure (seeFigure 3). The other two mutants P4 bp-1 cg andgc are readily suppressed by CYT-18, but show asigni®cant proportion of inactive RNA (42-48 %) inin vitro splicing assays (see above). We cannot dis-tinguish whether the second transition re¯ects dis-ruption of secondary or tertiary structure or both.The thermal denaturation analysis indicates thateven very small changes, such as substitution of adifferent base-pair at P4 bp-1 or P6 bp-1, stronglyaffect the folding of the group I intron catalyticcore. We could discern no systematic difference inmelting pro®les of mutants that could or could notbe rescued by CYT-18 (Table 4). Thus, the differ-ence must re¯ect that CYT-18 binding can promoteformation of the active tertiary structure in somemutant introns but not others.Fe(II)-EDTA analysisThe decreased amplitude of the tertiary structuretransition in the P4-P6 mutants could re¯ect eithera mixture of folded and unfolded RNAs or a uni-form population of RNAs that contains a subset oftertiary-structure interactions. To distinguish thesepossibilities, we carried out Fe(II)-EDTA structuremapping on several non-splicing P4-P6 doublemutants. These mutants are expected to have thegreatest structural disruptions, and two of them,cU/ug and uU/Gu, have the greatest ÁTm amongthose mutants that have single low temperaturetransitions (Table 4). Thus, we reasoned that anyresidual tertiary structure would have to formindependently of the correct geometry at the junc-tion of the P4-P6 stacked helices. As shown inFigure 6, the wild-type intron showed protections272 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  9. 9. throughout the P4-P6 and P3-P9 domains. By con-trast, the mutants lost most protections in the P4-P6 domain (e.g. P4, J4/5, P5, P6, and J6/6a), butretained protections in the P3-P9 domain (P7.2,P3[3H], P7, and P9) at both 3 mM and 8 mM Mg2‡.Together with the thermal denaturation analysis,these ®ndings indicate that the P4-P6 mutationsdisrupt tertiary structure that leads to protection ofthe P4-P6 domain, but that some regions of theintron remain at least partially folded.Equilibrium-binding assaysThe inability of CYT-18 to rescue some mutantscould re¯ect either that the folding defect is toogreat or that the CYT-18 binding site is impaired.To investigate how the mutations at the P4/P6junction affect the interaction with CYT-18, selectedmutant introns were used for protein-bindingassays. Equilibrium-binding experiments areshown in Figure 7, and the data are summarized inTable 4. For equilibrium binding, low concen-trations of 32P-labeled RNAs containing the coreregions of wild-type or mutant introns were incu-bated with increasing amounts of CYT-18, andbinding was assayed by retention of the labeledRNA on a nitrocellulose ®lter. The core regionRNAs were used so that the results could be com-pared more directly with those from RNA struc-ture mapping and thermal denaturation analysis.We con®rmed that the binding characteristics ofthe wild-type core region RNA are essentially thesame as that of the precursor RNA containing thefull-length ÁORF td intron and ¯anking exons (koffvalues ˆ 0.011 and 0.0086 sÀ1, respectively; Table 4and data not shown). The binding of CYT-18 to theFigure 5. UV-melting pro®les forwild-type and mutant td introns.RNAs corresponding to the coreregions of the wild-type andmutant td introns (0.2 A260;$0.32 mM) were heated slowlyfrom 25 C to 80 C, and A260 wasmonitored as a function of tem-perature. The plots show the ®rstderivative of the absorbance(dA260/dT) as a function of tem-perature (T). (a) P4 bp-1 purine-purine mutants. (b) P4 bp-1 base-paired and Ac mutants. (c) P6 bp-1purine-purine mutants. (d) P6 bp-1pyrimidine-pyrimidine (Pyr-Pyr)mutants. (e) P6 bp-1 base-pairedmutants. (f) P4-P6 double mutants.The melting pro®le for the wild-type td intron is included with eachgroup for comparison. Each assaywas repeated at least twice withsimilar results.Tyrosyl-tRNA Synthetase in Group I Intron Splicing 273
  10. 10. td intron is signi®cantly weaker than its binding tothe N. crassa mt introns (Kds 0.7 pM) and withina range where Kds can be measured directly byequilibrium methods (see Materials and Methods).The equilibrium binding assays show that wild-type td intron RNA binds CYT-18 with a Kd at37 C of 0.12 nM, with 19 % of the input RNAbound at saturating protein concentration. Therelatively small proportion of input RNA bound,which was unchanged using different renaturationprotocols, may re¯ect a combination of inef®cientretention of the complex on the nitrocellulose ®lter,the relatively rapid dissociation rate of CYT-18,and/or that some proportion of the RNA foldsinto an inactive conformation that is unable tobind CYT-18. Although the in vitro binding assaysmonitor only a proportion of the active RNAs,they nevertheless show a generally good corre-lation with the in vivo and in vitro splicing pheno-types (see below).For P4 bp-1, we analyzed CYT-18 binding of allmutants that have a purine-purine mismatch (Ag,gg, ga, and Aa), along with the four CYT-18 sup-pressible mutants that retain a base-pair at thisposition (cg, gc, gU and ug) and the mutant Ac. Inall these cases, the ability to bind CYT-18 mirroredthe ability of the protein to promote splicing.Among the purine-purine mismatches, the weakself-splicing mutant Aa bound CYT-18 relativelystrongly (Kd at 37 C ˆ 0.028 nM; 12 % bound), theCYT-18 suppressible mutant ga bound less well(Kd ˆ 2.5 nM, 12 % bound), and the two non-spli-cing mutants (Ag and gg) bound CYT-18 poorly(2.6 % bound) (Figure 7).The four CYT-18-suppressible mutants thatretain a base-pair at P4 bp-1 bound CYT-18 withKd values that were 17.5- to 75-fold higher thanthat of the wild-type intron (2.1 to 9.0 nM com-pared to 0.12 nM), with the weakest binding dis-played by the mutant ug, which is the leastTable 4. Thermal denaturation and CYT-18 binding properties of wild-type and selected mutant td intronsPhenotypebMutantaÀCYT-18 ‡CYT-18Tm(C)ÁTm(C)cKdd(% bound RNA)e(nM) kofff(sÀ1)Calculated kong(MÀ1sÀ1)WT ‡ ‡ ‡ ‡ ‡ ‡ 49.6 - 0.12 (19%) 0.011 9.2  107P4 bp-1 mutantsAa ‡ ‡‡ 50.3 0.7 0.028 (12%) 0.012 4.1  108Ag À À 42.0/53.9hÀ7.6/4.3 8.2 (2.5%) 0.011 1.3  106ga À ‡‡ 53.2 3.6 2.5 (12%) 0.0085 3.4  106gg À À 40.7/53.9hÀ8.9/4.3 3.8 (2.6%) 0.23 6.1  106cg À ‡ ‡ ‡ 40.7/53.9hÀ8.9/4.3 2.2 (10%) 0.012 5.5  106gc À ‡ ‡ ‡ 37.8/58.0hÀ11.8/8.4 2.1 (9.5%) 0.014 6.7  106gU À ‡ ‡ ‡ 48.7 À0.9 5.4 (13%) 0.015 2.8  106Ac À ‡ ‡ ‡ 50.6 1.0 0.46 (10%) 0.014 3.0  107P6 bp-1 mutantsaa À À 48.6 À1.0 (2%) À ‡ 49.0 À0.6 2.9 (3.9%) À ‡‡ 48.6 À1.0 1.2 (11%) À ‡‡ 50.3 0.7 1.0 (3.1%) À ‡ 49.6 0.0 (2%) À ‡ ‡ ‡ 49.6 0.0 0.11 (6.4%) 0.0087 7.9  107uc À ‡‡ 50.3 0.7 1.3 (3.5%) 0.0071 5.5  106uu À ‡ 50.6 1.0 9.4 (3.4%) 0.0090 9.6  105au ‡ ‡‡ 49.6 0.0 0.080 (21%) 0.0081 1.0  108cg ‡ ‡ ‡ ‡ 47.7 À1.9 1.5 (28%) 0.0063 4.2  106Gu À ‡‡ 50.6 1.0 0.29 (29%) 0.0079 2.7  107ua À ‡‡ 49.5 À0.1 0.67 (18%) 0.0076 1.1  107ug À ‡‡ 49.6 0.0 1.6 (12%) 0.010 6.7  106P4-P6 double mutantsAc/uC À À 48.7 À0.9 (2%) À À 47.3 À2.3 (2%) À À 50.4 0.8 (2%) À À 48.5 À1.1 (2%) À À 47.2 À2.4 (2%) and mutant td intron sequences are indicated as in Tables 1 and 3.bSplicing phenotypes are based on the data in Tables 1 and 3.cÁTm is calculated as ÁTm ˆ Tm (mutant) À Tm (WT).dKd, Equilibrium constants for binding of CYT-18 to the wild-type and mutant td introns. Values are averages for two or moredeterminations, with standard deviations less than 10 %.ePercentage of input RNA bound at saturating protein concentration.fkoff, Dissociation rate constants for the stable CYT-18/intron RNA complexes. Values are averages for two or more determina-tions, with standard deviations less than 10 %.gkon, Association rate constant calculated as kon ˆ koff/Kd.hSome mutants show two transitions below 60, not determined.274 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  11. 11. ef®ciently suppressed by CYT-18 (Kd ˆ 9.0 nM,6.7 % bound) (Figure 7(b)). The mutant Ac, whichis well suppressed by CYT-18, has a relatively lowKd value (0.46 nM) with $10 % of the RNA bound.We note that two of the three mutants that retainthe wild-type A residue at P4 bp-1[5H] have rela-tively low Kd values, the only exception being thenon-splicing mutant Ag, which forms an alterna-tive secondary structure. These ®ndings suggestthat the A residue at P4 bp-1 contributes directlyor indirectly to formation of the CYT-18 bindingsite. However, this contribution must be dispensa-ble, since an A is not found at this position in otherCYT-18-suppressible td intron mutants, nor in theN. crassa CYT-18-dependent introns (Wallweberet al., 1997).For P6 bp-1, we tested all purine-purine mis-matches, all pyrimidine-pyrimidine mismatches,and all mutants that retain a base-pair. Again,within groups of mutants, there was a good corre-spondence between CYT-18 binding and thedegree of suppression of the splicing defect.Among the purine-purine mismatches, the greatestextent of binding was displayed by mutant Ga,which is suppressed moderately by CYT-18(Kd ˆ 1.2 nM, 11 % bound) (Figure 7(c)). Themutant Gg, which is also moderately suppressed,binds with a lower Kd (1.0 nM), but only 3.1 % ofthe RNA was bound, suggesting that a smallerproportion of this intron can fold into the correctconformation. ag, which is weakly suppressed,showed weaker binding af®nity (Kd ˆ 2.9 nM,3.9 % bound), whereas the non-splicing mutant aa,showed little, if any, binding af®nity (2 % bound).Likewise for the P6 bp-1 pyrimidine-pyrimidinemismatches, the fully-rescueable mutant, cu,showed the best binding af®nity (Kd ˆ 0.11 nM,6.4 % bound), uC, which is moderately suppressed,showed intermediate binding (Kd ˆ 1.3 nM, 3.5 %bound), and the weakly rescued mutants, cC anduu, showed the weakest level of binding (2 %bound and Kd ˆ 9.4 nM, 3.4 % bound, respectively)(Figure 7(d)).As a group, the mutants that retain a base-pairat P6 bp-1 showed the highest proportion ofbound RNA (12 % to 29 %) (Figure 7(e)). All thesemutants are moderately (‡‡) or well (‡ ‡ ‡) sup-pressed by CYT-18. The mutants, au and Gu,which retain a purine at the 5Hposition of P6 bp-1,have Kd values comparable to wild-type (0.080 nMand 0.29 nM, respectively), while the other threemutants, cg, ua and ug, have somewhat increasedFigure 6. Fe(II)-EDTA cleavagepatterns of wild-type and selectednon-splicing P4-P6 mutant tdintrons. 5Hend-labeled in vitro tran-scripts containing wild-type ormutant td introns were incubatedwith (‡) or without (À) Fe(II)-EDTA in reaction media containing0, 3 or 8 mM Mg2‡, as indicated.Cleavage products were analyzedin a denaturing 9 % polyacrylamidegel, which was dried, autoradio-graphed, and quantitated with aphosphorImager. Wild-type (left)and mutant sequences (right) areshown above the lanes, with wild-type nucleotide residues indicatedin uppercase letters and mutantnucleotide residues indicated inlowercase letters. Ladders: OH,partial alkaline hydrolysis; G, par-tial hydrolysis with RNase T1; A,partial hydrolysis with RNase U2.Intron regions are demarcated tothe right. The open arrows indicateregions that are protected in bothwild-type and mutant RNAs, andthe ®lled arrows indicate regionsthat are protected in wild-type butnot in mutant RNAs.Tyrosyl-tRNA Synthetase in Group I Intron Splicing 275
  12. 12. Kd values (0.67 to 1.6 nM). The non-splicing doublemutants Ac/uC, cU/ug, cg/uC, gU/uC, and uU/Gu all showed little if any CYT-18 binding (2 % ofinput RNA bound), consistent with their inabilityto be suppressed by CYT-18 (Figure 7(f)). Acrossgroups, some mutants showed similar degrees ofCYT-18 binding, but different degrees of CYT-18suppression, suggesting that some mutations mayaffect steps after CYT-18 binding. Nevertheless, allP4 bp-1 or P6 bp-1 mutants that are non-splicingor weakly suppressed by CYT-18 showed at mostlow levels of CYT-18 binding.koff measurementsTo supplement the equilibrium-binding assays,we carried out koff measurements, which provideinformation about the strength of the complex onceit is formed. In these experiments, pre-formedCYT-18/intron RNA complexes were diluted intoreaction medium containing 1 mg/ml heparin, anddissociation of the complex was measured as afunction of time (Figure 8). As found previouslyfor other CYT-18-dependent group I introns(Wallweber et al., 1997), the dissociation curveswere biphasic with the initial fast phase represent-ing loosely associated protein and the second,slower phase representing the stable complex. Thefew cases where there appear to be discrepancieswith the proportion of bound RNA in the equili-brium binding assays (e.g. P4 bp-1 Ag, ga, and gg,and P6 bp-1 uC and uu) likely re¯ect differences inthe amount of loosely bound RNA for the differentmutants. The koff values for the stable complex aresummarized in Table 4. Since the Kd values weremeasured independently, the koff values could beFigure 7. Equilibrium-bindingassays for representative td intronmutants. 32P-labeled RNAs (4 pM)containing wild-type or mutant tdintrons were incubated withincreasing concentrations of CYT-18 protein dimer (0 pM-34 nM) for20 minutes at 37 C, and bindingwas assayed by the retention of the32P-labeled RNA on a nitrocellulose®lter. (a) P4 bp-1 purine-purinemutants. (b) P4 bp-1 base-pairedand Ac mutants. (c) P6 bp-1 pur-ine-purine mutants. (d) P6 bp-1pyrimidine-pyrimidine (Pyr-Pyr)mutants. (e) P6 bp-1 base-pairedmutants. (f) P4-P6 double mutants.Each assay was repeated at leasttwice with similar results.276 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  13. 13. used to calculate kon. Signi®cantly, the koff valuesfor all of the mutants were within a factor of twoof the wild-type value. Thus, for those mutantsthat show elevated Kd values, the decreased bind-ing appears to be due largely to substantiallyincreased kon values. Considered together with thestructural analysis, these ®ndings suggest that themutations cause structural disruptions that impairbinding of CYT-18. Once bound, however, CYT-18folds the intron into the required tertiary structure,and the resulting complex has a stability similar tothat of the complex with the wild-type intron.DiscussionOur results show that most mutations at thejunction of the P4-P6 stacked helices lead to loss ofself-splicing activity, but can be suppressed byCYT-18. Considering the P4 bp-1 and P6 bp-1mutants together, only four of 30 possible non-wild-type nucleotide combinations at either pos-ition retained any ability to splice in E. coli in theabsence of CYT-18, and even in these cases, spli-cing was not as ef®cient as for the wild-type intron(Table 1). Mutations at the P4-P6 junction that dis-rupt base-pairing or base-stacking interactions orthe base triple between P6 bp-1 and J3/4, presum-ably lead to altered geometry at the junction of theP4-P6 stacked helices. Thermal denaturation andFe(II)-EDTA analysis indicate that this altered geo-metry in turn leads to grossly impaired tertiary-structure formation. In the Fe(II)-EDTA analysis,P4-P6 double mutants, which are expected to havethe most severe structural disruptions, lost mostprotections in the P4-P6 domain, but retained someprotections indicative of tertiary-structure for-mation in the P3-P9 domain. The loss of protec-tions in the P4-P6 domain likely re¯ects loss ofinter-domain contacts, which cannot be madeproperly when the geometry at the junction of theP4-P6 stacked helix is distorted.The deleterious effects of nucleotide substi-tutions at the P4/P6 junction are consistent withcomparative sequence analysis, which shows thatP4 bp-1 is remarkably constrained, correspondingto either AU or UA in 98 % of all naturally occur-ring group I introns (Table 5). Among subgroup IAintrons, P4 bp-1 is UA in 85 % and AU in 12 %,including the td intron. Of the 15 mutant nucleo-tide combinations analyzed here, only Aa and uacould splice to some extent in the absence of CYT-18. In general, the constraints on P4 bp-1 couldre¯ect either a need for a speci®c geometry or ther-modynamic stability of this base-pair, or thatP4 bp-1 participates in an as yet unknown tertiaryinteraction, perhaps involving A263 in the 5Hstrandof P7, which is in proximity to P4 bp-1 in the struc-tural models (cf. Michel Westhof, 1990; Goldenet al., 1998). An additional possibility is that thestrong selection for either AU or UA re¯ects arequirement that the conformation of the base-pairbe isosteric on reversal. For an AU pair, this wouldrequire the adoption of a trans Watson-Crick con-®guration, with hydrogen bonds between the ade-nine N1 and uridine N3, and between the adenineN6 and uridine O2 (Leontis Westhof, 1998). ToFigure 8. koff measurements forrepresentative td intron mutants.32P-labeled RNAs (100 nM) contain-ing wild-type or mutant td intronswere incubated with 100 nM CYT-18 dimer in 100 ml TMKBDG bufferfor 30 minutes at 37 C, then mixedwith 900 ml of TMKBDG containing1 mg/ml heparin. At the indicatedtimes, 100 ml portions were with-drawn and the amount of complexremaining was assayed by nitrocel-lulose ®lter binding. (a) P4 bp-1purine-purine mutants. (b) P4 bp-1base-paired and Ac mutants. (c)P6 bp-1 pyrimidine-pyrimidine(Pyr-Pyr) mutants. (d) P6 bp-1base-paired mutants. Each assaywas repeated at least twice withsimilar results.Tyrosyl-tRNA Synthetase in Group I Intron Splicing 277
  14. 14. maintain such a con®guration, the adenosine sugarmoiety would have to adopt a syn conformation inorder to keep the local stands anti-parallel(Westhof, 1992). The effect would be to widen theminor groove in this region, perhaps facilitatingformation of the base-triple interaction betweenJ3/4 and P6 or some other interaction required forgroup I intron function.Our results indicate that P6 bp-1 is constrainednot only by its base-triple interaction with J3/4,but also by its base-stacking interaction withP4 bp-1. Comparative analysis con®rms thatP6 bp-1 covaries with P4 bp-1 (Gautheret et al.,1995). Among subgroup IA introns, only ®vecombinations have been found in nature, UA/GU(75 %), UA/GC (12 %), AU/GC (6 %), AU/GU(5 %), and UG/GC (2 %) (Table 5), with AU/GCfound in the td intron. Of the subgroup IA combi-nations tested here in the td intron, ua/GC splicesweakly in the absence of CYT-18, while ug/GC isnot self-splicing but is moderately suppressed byCYT-18 (Table 1). Other combinations that givesome self-splicing are Aa/GC and AU/au, whichare not found in naturally-occurring group Iintrons, and AU/cg, which is the predominantcombination in subgroup ID introns (Tables 1Aand B).Remarkably, CYT-18 can suppress mutationshaving nearly all possible combinations of nucleo-tides at either P4 bp-1 (13/15) or P6 bp-1 (14/15).These include mutations that affect base-pairing orbase-stacking interactions, as well as mutations atP6 bp-1 that affect formation of the normal basetriple with J3/4-3. The few non-suppressiblemutations at one or the other positions are all pur-ine-purine mismatches, and in the case of P4 bp-1,these lead to formation of an alternate, presumablynon-productive secondary structure. The ability ofCYT-18 to suppress nearly all nucleotide substi-tutions indicates that there are no indispensablebase-speci®c contacts between CYT-18 and P4 bp-1or P6 bp-1. This conclusion is consistent with stu-dies indicating that CYT-18 makes primarily phos-phodiester backbone contacts in this region (seeIntroduction). Mutants that retain the wild-type A-residue at P4 bp-1[5H] and purine at P6 bp-1 havelower Kd values, but the effect is due primarily toan increased kon rather than increased stability ofthe complex. Although it remains possible thatbase-speci®c contacts make some contribution,these ®ndings suggest that the preferred nucleo-tides act primarily by making it easier for theintron to fold into the productive RNA structure.In most cases, suppression of the splicing defectby CYT-18 correlates with its ability to bind to themutant intron. The CYT-18-suppressible intronsbind the protein with Kd values up to 79-fold high-er than that for the wild-type intron, but in allcases the koff for the complex remains within two-fold of the wild-type value, and the increased Kdre¯ects primarily an increased kon for the bindingof CYT-18 to the misfolded intron. These ®ndingssuggest that the CYT-18 binding site is not formedproperly in the misfolded intron, but is induced byTable 5. Base-pair frequencies at P4 bp-1 and P6 bp-1 in naturally occurring group I intronsBase-pair frequenciesc(%)SubgroupaNumber ofsequencesbGC CG UA AU GU UG AA AC CA UC UUP4 bp-1A 87 85 12 2 1B 117 85 7 2 1 5C1-2 318 99 0.5 0.5C3 321 98 2D 17 100E 59 32 68P6 bp-1A 87 20 1 79B 117 79 2 4 13 2C1-2 318 0.5 5 1 92 1.5C3 321 1 99D 17 12 76 6 6E 59 62 2 36UA/GU UA/GC AU/GC AU/GU AU/CG UA/UA UG/GC OthersdP4-P6 combinationsA 87 75 12 6 5 2B 117 9 73 4 14C1-2 318 92 5 3C3 321 97 3D 17 12 76 12E 59 25 5 56 10 4aIntron classi®cation based on Michel Westhof (1990) and Suh et al. (1999).bThe number of group I intron sequences in each subgroup publically available as of January 2000.cPercentage of introns having indicated combinations. Combinations not found in naturally occurring group I introns are notlisted.dAll other combinations.278 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  15. 15. binding of CYT-18, so that the resulting complexhas nearly the same stability as the wild-type com-plex. Since the primary high-af®nity binding sitefor CYT-18 is around the junction of the P4-P6stacked helices (see Introduction), the most likelypossibility is that CYT-18 binds initially to thisregion to induce formation of the correct geometryat the junction of the P4-P6 stacked helix. Theresulting complex can then form the requiredscaffold for RNA and protein interactions with theP3-P9 domain.Those mutants that cannot be suppressed byCYT-18 presumably have structural defects thatmake it dif®cult for CYT-18 to induce the correctgeometry at the junction of the P4-P6 stackedhelices. The analysis of double mutants at P4 bp-1and P6 bp-1 shows that CYT-18 has dif®culty sup-pressing mutations that simultaneously affect bothbase-pairing and base-stacking interactions, par-ticularly when P6 bp-1 is unpaired. A predictionfrom our results is that naturally occurring group Iintrons that have disfavored nucleotide combi-nations at the P4-P6 junction may be non-self-spli-cing, protein-dependent introns. Althoughbiochemical data are lacking for most introns inthe database, two sea anemone mtDNA group Iintrons (Genebank accession numbers U36783 andU36784), which have a UC mismatch at P4 bp-1,were found to be non-self-splicing in vitro (Beagleyet al., 1996), and the Saccharomyces douglasii aI1intron (Genebank accession number M97514),which also has a UC mismatch at P4 bp-1 alsoappears to be protein dependent (Tian et al., 1993).Furthermore, consistent with our ®ndings that pur-ine-purine mismatches at P4 bp-1 or P6 bp-1impede splicing, GA, GG, and AG are not found ateither position in naturally-occurring group Iintrons, while AA, which we ®nd to be a relativelypermissible substitution only at P4 bp-1, is presentat P4 bp-1 in two group IB introns but is not foundat P6 bp-1 (Table 5).Together with previous work, our ®ndings forthe suppression of td intron mutants suggest amodel in which CYT-18 contacts both sides of theP4-P6 stacked helix and promotes formation of thecorrect geometry of the phosphodiester backbonein this region. Although the CYT-18 binding sitemay be preformed in the wild-type td intron,which is self-splicing, the same induced-®t modellikely applies to the N. crassa mt introns, which arenot self-splicing and do not fold into the catalyti-cally active RNA tertiary structure in the absenceof CYT-18 (see Introduction). Indeed, recent studiesdirectly demonstrate a CYT-18-induced confor-mational change in the isolated P4-P6 domain ofthe N. crassa mt LSU intron (M.G. Caprara A.M.L., unpublished results). Finally, we notedthat the P4-P6 stacked helix is structurally analo-gous to the anticodon arm/D-arm stacked helix oftRNATyr, which is recognized by the C-terminaltRNA-binding domain of bacterial TyrRSs(Bedouelle et al., 1993). The ability of CYT-18 toinduce the correct structure of the P4-P6 domaincould re¯ect that it uses the same binding siteadapted to recognize the phosphodiester backboneconformation of the anticodon/D-arm stackedhelix. This structure may be conformationally rigidin the tRNA, but can be induced in the group Iintron mutants studied here.Materials and MethodsE. coli strains and growth mediaE. coli strain 1904, a thyA::Kanrderivative of C600(Bell-Pederson et al., 1991), was used for td assays.DH5aFHwas used for plasmid ampli®cation and cloning,and BL21(DE3; plysS) was used for expression of CYT-18protein. Bacteria were grown in LB or minimal medium(MM) supplemented with 0.1 % Norit-A-treated Casami-no acids and 0.2 % (w/v) glucose (Belfort et al., 1983).Thymine was added at 50 mg/l. Antibiotics were addedat the following concentrations: ampicillin, 100 mg/l;kanamycin, 40 mg/l; chloramphenicol, 25 mg/l; and tri-methoprim, 20 mg/l.Construction of td intron mutantsThe wild-type construct, pTZtd1304 (Myers et al.,1996), contains a 265 nt ÁORF-derivative of the full-length td intron (Galloway Salvo et al., 1990), withadditional sequence changes U34A and U976G that cre-ate SpeI and EcoRI sites, respectively, for the introductionof mutations in the P4-P6 region. pTZtdÁP4-7, used as anegative control, has an internal deletion (Á69-875) inthe intron core and does not splice in the presence orabsence of CYT-18 protein (Mohr et al., 1992).The P4 bp-1 mutants were generated via two overlap-ping primers, TDP4 bp-1A (5HTTA TAC TAG TAA TCTATC TAA NCG GGG AAC CTC TCT AGT AGA C 3H)and TDP4 bp-1B (5HGAA CCC GGG CAG TCC TACAAT TTA GCN CGG GAT TGT CTA CTA GAG AGG3H). First, the primers were annealed and ®lled in withphage T7 DNA polymerase to generate a double-stranded DNA fragment that corresponds to intron pos-itions 27 through to 854 and covers the SpeI and SmaIsites in the intron. This segment was then ampli®ed byPCR with primers XC1 (5HACT TAT ACT AGT AAT C3H) and XC2 (5HGAA CCC GGG CAG TCC TAC 3H) (25cycles, 30 seconds at 94 C, 30 seconds at 55 C and 90seconds at 72 C, followed by extension for ten minutesat 72 C). The resulting PCR product was puri®ed in a1 % agarose gel, digested with SpeI and SmaI, and clonedbetween the corresponding sites of pTZtd1304 (Figure 1).To construct the P6 bp-1 mutants, two overlappingsegments of the intron were generated by PCR usingpTZtd1304 as template with primers XC5 (5HGAC TGCCCG GGT TCT ACA TAA ATG NCT AAC G 3H) plusNBS2 (5HGAC GCA ATA TTA AAC GGT 3H), and XC6(5HAAC CCG GGC AGT CCT ACA ATT TAG NACGGG T 3H) plus YK2 (5HGTA GAT GTT TTC TTG GGT3H). The PCR products were puri®ed in a 1 % agarose gel,and 0.1 mg of each independently ampli®ed segment wasmixed, and ampli®ed in a single PCR reaction with theoutside primers YK2 and NBS2. The PCR product wasgel-puri®ed, digested with EcoRI and SpeI, and clonedbetween the corresponding sites of pTZtd1304 (Figure 1).To construct the P4-P6 double mutants, PCR was car-ried out using pTZtd1304 as template with the junctionprimer (5HTTA TAC TAG TAA TCT ATC TAA NCGGGG AAC CTC TCT AGT AGA CAA TCC CGN NCTTyrosyl-tRNA Synthetase in Group I Intron Splicing 279
  16. 16. AAA TTG TAG GAC TGC CCG GGT TCT ACA TAAATG NCT AAC GAC TAT CCC T 3H) and reverse primer(5HCTC TGC GCG CAG CTG CCA GT 3H). The PCR pro-duct was then digested with EcoRI and SpeI and clonedbetween the corresponding sites of pTZtd1304.Colony assays of CYT-18-dependent splicing ofmutant td intronstd plating assays were carried out as described (Mohret al., 1992; Myers et al., 1996). Libraries of pTZ plasmidscontaining mutant td introns were transformed intoE. coli DH5aFH. Plasmid DNAs were isolated from ran-domly chosen colonies and transformed into E. coli 1904(C600 thyA::Kanr) in combination with either the vectorpACYC184 (Chang Cohen, 1978) or with pA550,which expresses the wild-type CYT-18 protein (Mohret al., 1992). Cells were grown overnight at 37 C on LBplates containing kanamycin for selection of the thyAÀhost strain, ampicillin for selection of the pTZtd plasmid,and chloramphenicol for selection of pA550 orpACYC184. For plating assays, 1 ml of cells were spottedon plates containing either minimal medium (MM), mini-mal medium plus thymine (MMT), or minimal mediumplus thymine plus trimethoprim (TTM), in the presenceof the above antibiotics. Splicing phenotypes werecharacterized after overnight incubation at 37 C and arebased on analysis of four to ten colonies in at least twoindependent experiments (Mohr et al., 1992). Phenotypesare de®ned as follows: ‡ ‡ ‡ , maximal splicing, growthon MM comparable to MMT and no growth on TTM;‡ ‡ , moderate splicing, growth on MM comparable toMMT and incomplete inhibition on TTM; ‡, weak spli-cing, growth on MM less than or equal to growth onMMT and growth on TTM comparable to MMT; À, nosplicing, no growth on MM and growth on TTM com-parable to MMT. After scoring the phenotypes,mutations in the targeted regions were identi®ed bysequencing using primers YK2 or NBS2 (see above). Themutant td introns were then sequenced completely toinsure that no adventitious mutations had been intro-duced during the constructions.In vitro transcriptionIn vitro transcription was for two hours at 37 C in100 ml of reaction medium containing 500 units phage T7RNA polymerase (Life Technologies, Gaithersburg, MD),2 to 5 mg of DNA template, 1 mM of each rNTP, 40 mMTris-HCl (pH 8.0), 25 mM NaCl, 8 mM MgCl2, 2 mMspermidine-(HCl)3, 5 mM dithiothreitol (DTT), 0.1 mCi[a-32P]UTP (3000 Ci/mmole; New England Nuclear, Bos-ton, MA) and 1 ml human placental ribonuclease inhibi-tor (Amersham, Arlington Heights, IL). 32P-labeledtranscripts used in in vitro splicing experiments and 5Hend-labeled transcripts used in Fe(II)-EDTA cleavageexperiments were synthesized as described (Myers et al.,1996). Higher speci®c activity 32P-labeled transcriptsused in equilibrium binding and kon experiments weresynthesized in a reaction mixture containing 0.2 mMATP, GTP, CTP, 0.02 mM UTP, and 0.35 mCi[a-32P]UTP. RNA transcripts for in vitro splicing weregenerated from PCR-ampli®ed DNA templates asdescribed (Myers et al., 1996). Unlabeled transcripts usedfor thermal denaturation experiments were synthesizedfrom gel-puri®ed, PCR-generated DNA templates usinga MEGAscriptTMkit (Ambion, Inc., Austin TX).After transcription, the DNA template was digestedwith DNase I (Pharmacia, Piscataway, NJ; 50 units for 20minutes at 37 C), and the transcripts were extractedwith phenol-CIA. Unlabeled transcripts used in thermaldenaturation experiments and 32P-labeled transcriptsused in Fe(II)-EDTA experiments and in vitro splicingexperiments were puri®ed in denaturing 4 or 6 % poly-acrylamide gels, ethanol precipitated, dissolved in dis-tilled water, and centrifuged (3000 g, four minutes 20 C)through a Sephadex G-50 spun column (Sigma, St. Louis,MO). Other 32P-labeled transcripts were centrifugedthrough a Sephadex G-50 spun column and ethanol pre-cipitated. The quality of transcripts was monitored bygel electrophoresis. Prior to use, all transcripts wererenatured by heating to 60 C in reaction medium with-out Mg2‡for ten minutes, then adding MgCl2 to give thedesired Mg2‡concentration and slowly cooling to roomtemperature. This renaturation protocol was found togive maximal extents of self-splicing for the wild-typetd intron construct pTZtd1304 compared to otherrenaturation protocols.In vitro splicing assaysIn vitro splicing reactions were carried outas described by Myers et al. (1996). Brie¯y, renatured,32P-labeled precursor RNA was preincubated with orwithout 200 nM CYT-18 protein dimer in the splicingreaction medium (100 mM KCl, 3 mM MgCl2, 20 mMTris-HCl (pH 7.5)) on ice for 30 to 45 minutes, thenwarmed to 37 C before initiating splicing by addition of5 mM prewarmed GTP. The reaction was incubated at37 C, and time points were taken at different intervalsfrom 0 minutes to two hours. The products wereanalyzed in a denaturing 6 % polyacrylamide gel, whichwas dried and quantitated with a phosphorImager.Kinetic parameters were calculated as described (Myerset al., 1996).RNA structure mappingRNA structure mapping experiments with DMS andFe(II)-EDTA were as described (Caprara et al., 1996a;Myers et al., 1996). DNA templates used to generatein vitro transcripts were derived from the correspondingwild-type or mutant pTZtd plasmids by PCR with pri-mers TDSXM5H(5HGGT ACC TAA TAC GAC TCA CTATAG GGC CTG AGT ATA AGG TGA C 3H), which con-tains a sequence corresponding to intron positions 8 to26 and adds a phage T7 promoter, and TDSXM3H(5HTTTAAA TGT TCA GAT AAG GTC GTT AA 3H), which con-tains a sequence complementary to intron positions 992-1012. Transcription with phage T7 RNA polymeraseyields 260 nt RNAs that begin at intron position G8 andend at A1012, 5 nt upstream of the 3Hsplice site. Thetranscripts have an extra 5HG-residue derived from theT7 promoter, and 5 extra nt (5HAAUUU) at their 3Hendderived from the PCR primer used to generate the DNAtemplate.Thermal denaturation analysis of mutant td intronsThermal denaturation analysis was as described byBrion et al. (1999b). DNA templates used to obtain in vitrotranscripts were generated by PCR of wild-type ormutant pTZtd plasmids using the primers XC19 (5HTGTTCA GAT AAG GTC GT 3H), which is complementary tointron positions 995-1011, and one of four primers280 Tyrosyl-tRNA Synthetase in Group I Intron Splicing
  17. 17. XC18A, XC18G, XC18C and XC18T (5HATT TAA TACGAC TCA CAT TAG AAT CTA TCT AAN CGG 3H) thatcontain a T7 promoter sequence and a variable nucleo-tide N that depends on the identity of the nucleotide atP4 bp-1 in the mutant being studied. Transcription withT7 RNA polymerase yields 224 nt RNAs correspondingto the catalytic core of the ÁORF intron from positionA37 in J2/3 to A1011 in J9.2/10, with an extra 5HG resi-due from in vitro transcription. The wild-type transcriptis identical to that used by Brion et al. (1999b), except forthe mutation U976G introduced to create an EcoRI sitefor cloning.Thermal denaturation was carried out in a Perkin-Elmer Bio20 UV/Vis spectrometer equipped with water-thermostatable automatic six-cell changer controlled by aPTP-6 temperature controller (Perkin-Elmer). RNA (0.2A260, $0.32 mM) was dissolved in 3 ml of reaction med-ium (50 mM Na cacodylate (pH 7.5), 50 mM NH4Cl,3 mM MgCl2 ) and renatured by heating to 60 C for ®veminutes and then slowly cooled to room temperature.After de-gassing, A260 was measured as a function oftemperature from 25 C to 80 C, with a heating rate of0.2 C/minute. Temperature was monitored by an in-sample digital temperature sensor (Perkin-Elmer), whichhad been calibrated with a digital circulation water bath,as well as a thermometer. Plots of the ®rst derivativeof A260 versus temperature were generated using theTempLab software provided by Perkin-Elmer.Equilibrium bindingRNA binding assays were carried out with the samein vitro transcripts used for RNA structure mappingexperiments (see above). For equilibrium binding assays,4 pM 32P-labeled RNA (11,500 Ci/mmol) was incubatedwith increasing amounts of CYT-18 protein in 500 ml ofTMKBDG buffer (10 mM Tris-HCl (pH 7.5), 5 mMMgCl2, 100 mM KCl, 100 mg/ml acetylated bovine serumalbumin, 5 mM dithiothreitol and 10 % (v/v) glycerol)for 20 minutes at 37 C, then quickly mixed (5 seconds)with an equal volume of pre-warmed reaction mediumcontaining 2 mg/ml heparin and ®ltered through nitro-cellulose prewetted with TMK (10 mM Tris-HCl (pH 7.5),5 mM MgCl2, 100 mM KCl). The ®lters were washedthree times with 1 ml of TMK buffer, dried, and countedusing a Beckman LS6500 scintillation counter (BeckmanInstruments, Fullerton, CA). Since the 20 minute equili-bration time is more than ten half-lives of the RNA-pro-tein complex, 99.9 % of the complexes formed shouldhave reached equilibrium. The brief (5 seconds) incu-bation with heparin was found to be essential to elimin-ate non-speci®c binding at high CYT-18 concentrations.Based on the measured koff values (Table 4), 5 % ofthe speci®c complex with the wild-type intron(t1/2 ˆ 62.2 seconds) would dissociate during the heparinincubation, and 11 % of the least stable of the com-plexes with the mutant introns (Table 4). Data were ®t tothe equation ([P] ‡ [R] ‡ Kd)/2 À (([P] ‡ [R] ‡ Kd)2/4 À [P].[R])1/2, where [P] is the concentration of CYT-18dimer used in the experiment and [R] represents pMRNA bound by CYT-18 at the saturating protein concen-tration.koff measurementsThe dissociation rate constants (koff values) of theCYT-18/intron RNA complex were measured asdescribed by Wallweber et al. (1997), with minor modi®-cations. Brie¯y, complexes were formed by incubating100 nM CYT-18 dimer with 100 nM 32P-labeled RNA in100 ml of TMKBDG buffer (see above) for 20 minutes at37 C. The complexes were then mixed with 900 ml ofTMKBDG containing 1 mg/ml heparin to bind unasso-ciated CYT-18 protein. At different times, 100 ml aliquotswere removed and the amount of complex remainingwas assayed by nitrocellulose ®lter binding, as describedabove. Data were ®t to an equation with two exponen-tials, A1(eÀk1t) ‡ A2(eÀk2t), where k1 and k2 are the koffvalues for the stable and rapidly dissociating RNP com-plex, and A1 and A2 are the amplitudes, representing theproportion of RNA in each complex.Comparative sequence analysis of the group I intronP4-P6 stacked helical junctionComparative sequence analysis was performed asdescribed (Gautheret et al., 1995) using a database, whichas of January 2000, contained more than 900 group Iintron sequences assigned to the major structural sub-groups A, B, C1-2, C3, D and E (Michel Westhof, 1990;Suh et al., 1999). Within each subgroup, group I intronsequences were aligned manually with the program AE2(T. 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