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2 Proc. Nti. Acd. Sci. USA Vol. 76, No. 7, pp , July 1979 Biochemistry Inititor trnas hve unique nticodon loop conformtion (S1 endonuclese/stem-loop interction/conformtionl probe/chin elongtion trna) PAUL WREDE, NANCY H. WOO, AND ALEXANDER RICH Deprtment of Biology, Msschusetts Institute of Technology, Cmbridge, Msschusetts 2139 Contributed by Alexnder Rich, My 3,1979 ABSTRACT Trnsfer RNA (trna) molecules hve been lbeled with 32P t the 5' end nd subjected to SI nuclese digestion. The products were nlyed by high-resolution gel electrophoresis. Three inititor trnas nd six chin-elongting trnas were exmined. SI nuclese cleved Escherichi coli trnafmet, yest trnafmet, nd mmmlin trnafmet t the sme two positions in the nticodon loop. In contrst, SI nuclese cleved the nticodon loop of E. coli trnammet, yest trna Met, yest trnaphe, Schioscchromyces pombe trna~ ', E. coli trna2giu, nd E. coli trnatrp (su+) t four positions generlly, except where modified nucleotide in the wobble position inhibited the enyme. The mrked contrst between these clevge ptterns suggests different conformtion for the nticodon loops of these two clsses of trna molecules. It is suggested tht the specilied conformtion in the nticodon loop of inititor trnas my be due to specil sequence of GC bse pirs in the djoining nticodon stem. One of the interesting fetures of the genetic code is the fct tht one codon, AUG, hs two distinct functions. It codes for the methionine residues tht initite protein synthesis in both prokryotic nd eukryotic systems nd it lso codes for methionine residues tht re internl in the polypeptide chin sequence. These distinct functions re crried out by two different trna isocceptors. One of these is the inititor trna (trnafmet), which cts solely to initite polypeptide chin The publiction costs of this rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U. S. C solely to indicte this fct. synthesis, while the other (trnammet) is used for internl methionine positions. Both of these trnas for methionine hve the nticodon CAU, which is complementry to the methionine codon AUG. However, wht distinguishes n inititor trnafmet from chin elongting trnammet is uncler. Considerble informtion hs ccumulted regrding the nucleotide sequence of these two clsses of trna coding for methionine (1). The results re fr from instructive. The mjor prokryotic difference is the lck of one complementry bse pir on the cceptor stem of the inititor in comprison to chin-elongting trnas (see Fig. 1 A nd D). Severl investigtors hve pointed out tht there re unusul sequences ssocited with eukryotic inititor trnas (2-4). In lower eukryote, such s yest, the fmilir sequence in the T-loop, T-4/-C-G, is replced by the sequence A-U-C-G (Fig. 1B). However, the trna cceptor stem hs norml complement of seven bse pirs. In higher eukryotes, such s mmmls, the inititor trna contins the sequence A-U-C-G in the T-loop but hs n dditionl puling substitution of cytosine for urcil in position 33, which is t the 5' side of the nticodon in ll trnas (Fig. 1C). A urcil residue is found there in ll other trnas. There ppers to be no simple correltion between nucleotide sequence nd inititor functions. Nonetheless, inititor trna molecules form functionlly distinct clss; in mny instnces one inititor cn be substituted for nother in n in vitro protein synthetic system (5). The principle difference between n inititor trna nd chin-elongting trnas is the bility of the former to bind t the ribosoml P site nd thus initite polypeptide chin synthesis while the ltter bind initilly only t the ribosoml A site (6). In n ttempt to understnd so'me spects of this system, we hve crried out experirnents in which both inititor nd chin-elongting trna molecules hve been subjected to nucleolytic clevge by the endonuclese SI from Aspergillus orye. This nuclese is strict single-strnd-specific endonuclese which hs been used in number of studies s probe of secondry structure, specificlly, to detect loop regions in DNA s well s RNA (7-11). Here we report tht despite considerble differences in the nucleotide sequences of prokryotic nd eukryotic inititor trnas, there is common SI nuclese digestion pttern for the nticodon loop for inititor trnas. Furthermore, this pttern is quite different from tht seen in chin-elongting trnas, including the trnas tht code for internl methionine residues in protein biosynthesis. These findings suggest tht the nticodon loop conformtion of inititor trnas, s clss, differs from tht of chin-elongting trnas in mnner tht is detected by the structurlly sensitive Si nuclese probe. MATERIALS AND METHODS Escherichi coli nd humn plcent inititor trna were gift from B. A. Roe. E. coli trnammet ws donted by L. H. Schulmn. The yest inititor trna ws gift from U. L. RjBhndry. Yest trnammet ws gift from H. Feldmnn (Munich, West Germny). Schioscchromyces pombe trnaphe ws gift of D. Soll nd E. coli trnatrp (CA 16) ws gift of R. Buckinghm (Pris). A. orye SI nuclese ws purchsed from Boehringer Mnnheim, nd purified preprtion ws provided by J. Vournkis. The commercil Si nuclese ws found to be contminted by trce mounts of T1 nuclese ctivity. However, comprison of the results obtined with this enyme preprtion with those given by the enyme furnished by J. Vournkis did not show significnt differences in the nticodon loop clevges. All the trnas exmined were lbeled t the 5' end with 32p by stndrd techniques (8, 9). After digestion, the results were nlyed by modifiction of the rpid gel sequencing method (12) described by Wurst et l. (8) nd Wrede et l. (9). Nucleotide clevge sites were identified by comprison with "ldder" produced by lkline hydrolysis, in conjunction with vrious sequence-specific enymtic clevges. The lkline hydrolysis ws performed in 5 mm NOH/1 mm EDTA t 1 C for 2 sec. The sequence-specific nucleses used to ssign the lkline hydrolysis frgments were Ti nuclese from A. orye, pncretic RNse A (both purchsed from Boehringer Mnnheim), nd Physrum I nuclese ( gift from H. Donis- Keller). All Si nuclese digestions were performed in stndrd buffer contining 25 mm NOAc (ph 4.5), 5 mm MgCl2, 5 mm KC1, nd 1 mm Zn(OAc)2, t 37 C, except where specifi- 3289

3 Biochemistry: Wrede et l. clly noted. In order to determine precisely the initil clevge sites, the digestions of ech trna were crried out for vrying intervls s noted in Fig. 2. The rections were stopped by djusting the rection mixture to 4 mm ATP (ph 7.). All trnas were preincubted in the rection mixture for 1 min t 37C prior to ddition of the enymes. The trna numbering system used here is tht dopted by Guss et l. (1) in their colltion of trna sequences. In this system, the constnt uridine in the nticodon loop is lbeled s US3 irrespective of the totl number of nucleotides in prticulr trna sequence (see Fig. 1). It is importnt to note tht Si nuclese digestion frgments terminte in 3'-hydroxyl group while lkline hydrolysis frgments hve 3'-phosphte. Therefore, Si nuclese frgments migrte slightly more slowly thn the corresponding lkline hydrolysis frgments on sequencing gels. In prticulr, the bnds of Si nuclese frgments often hve position between two djcent bnds of lkline hydrolysis frgments. In order to ssign uniquely the 3' bse of Si frgments, we hve used sequence-specific nucleses nmely, pncretic RNse A, Physrum I nuclese, nd T1 nuclese. A complete description of the procedure we hve devised to unmbiguously nlye SI nuclese clevge sites is in preprtion. RESULTS The method used to estblish clevge sites by Si nuclese involved n initil lbeling of the trna 5' end with 32P nd subsequent chemicl nd enymtic clevge of the molecule, which ws nlyed by high-resolution crylmide gel electrophoresis. The results of study on E. coli trnafmet is shown in Fig. 2. Ten lnes were run in prllel, which provided the informtion needed to identify the Si clevge sites in the nticodon loop. Alkline hydrolysis produced complete ldder Proc. Ntl. Acd. Sci. USA 76 (1979) 3291 of frgments, the identity of which cn be determined most directly through the use of A. orye endonuclese T1, which cleves fter gunosine residues, nd Physrum I nuclese, which cleves predominntly fter denine nd urcil residues. These two enymtic digestions were performed in the presence of 7 M ure, which dentures the trna but does not inhibit either enyme. The combintion of these results (lnes 2 nd 3, Fig. 2) llows one to ssign numbering system to the lkline hydrolysis ldder (lnes 5 nd 1). Prtil S1 nuclese digestions under nondenturing conditions re shown in lnes 4, 6, nd 8 in Fig. 2. Vrious incubtion times were used in order to determine the initil clevge site. S1 nuclese produces two principle clevge sites. The precise identifiction of these clevge sites is complicted by the fct tht S1 nuclese frgments hve 3'-hydroxyl group while lkline hydrolysis frgments terminte in 3'-phosphte group. Consequently, S1 nuclese frgments migrte more slowly thn the corresponding lkline hydrolysis frgments. Pncretic RNse A ws used under nondenturing conditions to fcilitte the identifiction of the 3' terminus of S1 nuclese frgments (lne 7, Fig. 2). Pncretic RNse A cleves fter pyrimidines, especilly those 5' to n denosine, yielding frgments with 3'-phosphte. The digestion shows two clevge sites, C34 nd U36, in the nticodon loop. A comprison of the two S1 nuclese bnds with the pncretic RNse bnds llows one to mke definite ssignment of C34 nd AS5 to the S1 nuclese bnds. The S1 nuclese digestion in Fig. 2, lnes 4, 6, nd 8, shows the clevge fter 2 min, 5 min, nd 3 min, respectively. The reltive intensity of the two bnds in the nticodon region remins bout the sme over this extended digestion period. Further, one does not see the introduction of new clevge sites in the nticodon loop over this time period. The S1 digestion , qw,,,, C28 - C25 -G22 -A w -U1 7c d* -C16 A35 S- sc M A35 -C34-3 I.. ~~U36 ~~~C334 *4**...U3 C36 U35 U33 UMN b c ci e FIG. 2. Autordiogrms of prtil S1 nuclese digestions of 5'-lbeled [32P]tRNAs. The sequencing lnes re shown only in for E. coli trnafmet. All rection mixtures contined the stndrd buffer except when noted. S1 nuclese digestions were crried out t.5 unit/pg of trna t 37 C for vrying time periods. All lkline hydrolysis (NOH) re s described in Mterils nd Methods. () E. coli trnafmet: (Lne 1) no enymes dded, 3 min, 37 C; (lne 2) T1 nuclese in 7 M ure/1 mm EDTA,.5 unit of enyme per ug of trna, 62 C, 3 min; (lne 3) sme s lne 2 except Physrum I nuclese ws used; (lne 4) S1 nuclese, 2 min; (lne 5) NOH; (lne 6) S1 nuclese, 5 min; (lne 7) pncretic RNse A, 4 X 1-4 unit/,pg of trna, 1 min, 37 C; (lne 8) S1 nuclese, 3 min; (lne 9) sme s lne 3; (lne 1) NOH. (b) Yest trnafmet: (Lne 1) no enyme dded; (lnes 2-4) Si nuclese t 1 sec, 3 sec, nd 1 min, respectively; (lne 5) NOH; (lne 6) no S1 nuclese dded, 3 min. (c) Humn plcent trnafmet: (Lne 1) no enyme dded, 3 min; (lnes 2 nd 3) S1 nuclese, t 1 min nd 5 min, respectively; (lnes 4 nd 7) NOH; (lnes 5 nd 6) S1 nuclese t 1 min nd 3 sec, respectively. (d) Yest trnammet: (Lne 1) no enyme; (lne 2) NOH; (lnes 3, 4, nd 5) S1 nuclese t 1 hr, 2 min, nd 1 min, respectively. (e) E. coli trna2glu: (Lne 1) no enyme dded, 3 min; (lne 2) NOH; (lne 3) S1 nuclese, 5 min. Ten microgrms of trna were used in ech slot except in the experiments with yest trnammet, where 2,g/slot were used. All digestions were run on 2% polycrylmide gel/7 M ure s described (9).

4 3292 Biochemistry: Wrede et l. lnes lso show tht wek clevge sites exist t G23, G2, nd Gi9. These re due to smll mount of contminting Ti nuclese ctivity in the commercil Si nuclese; purified SI nuclese preprtions did not show these dditionl Ti clevge sites. The diffuse bnds ner the top of the gel in the SI lnes re found in lne 1, where no enyme ws dded. Thus, the only clevges produced by Si nuclese digestion in E. coli trnafmet re the two found in the nticodon loop. A study of the type shown for E. coli trnafmet ws crried out on severl other trna molecules. Some of the results re shown for yest trnafmet (Fig. 2b), mmmlin trnafmet (Fig. 2c), yest trnammet (Fig. 2d), nd E. coli trna2glu (Fig. 2e). In Fig. 2b, the prtil Si nuclese digestion of yest trnafmet shows two prominent clevge sites in the nticodon loop nd two slightly weker sites in the T-loop. In contrst to the results with E. coli trnafmet, the lower clevge site, identified s C34, is somewht more intense thn the clevge t AS5. However, the two bnds pper simultneously in the kinetic experiment. The results with the mmmlin trnafmet (Fig. 2c) show two Si nuclese clevge sites in the nticodon loop. Agin, these hve been identified s C34 nd AS5. Both sites pper to be eqully susceptible since they hve pproximtely identicl intensities for ll of the digestion intervls tken. The results of prtil Si nuclese digestion on yest trnammet re shown in Fig. 2d. In contrst to the inititor trna digestion pttern, there re four clevge sites in the nticodon loop. The identifiction of the four clevge sites cn be mde by compring the Si nuclese bnds with the lkline hydrolysis ldder shown in lne 2. A similr pttern is seen in Fig. 2e, which shows the results of S1 nuclese digestion of E. coli trna2glu. The nticodon loop contins four clevge sites. Results similr to these hve been presented in refs. 8 nd 9. In both trnas, the clevge sites re residues 33, 34, 35, nd 36, with the initil clevge sites being 34, 35, nd 36 s determined kineticlly. We hve crried out similr experiments on other trnas. These include E. coli trname, yest trna h, Schio. pmbe trnaphe, nd E. coli trnatrp (su+). In ech of these trnas, positions 33, 35, nd 36 were found to be S1 nuclese clevge sites. Residue 34 ws lso clevge site for the E. coil trnatrp. The lck of clevge by SI nuclese t this position in the two phenyllnine trnas is undoubtedly the result of Si nuclese inhibition by the 2'-methyl group of Gm34. Modifictions in the ribose t this position inhibit SI nuclese (8, 9). Likewise, Tble 1. Proc. Ntl. Acd. Sci. USA 76 (1979) the lck of clevge t c4c34 in E. coli trnammet probbly reflects inhibition of the enyme due to the modifiction of the bse. In ll these trnas, the clevge t U33 ppers to be secondry clevge site. The results of Si nuclese digestion on ll the trnas tested re summried in Tble 1. Similr results were lso obtined with digestion t ph 6.. DISCUSSION The results of this investigtion, summried in Fig. 1 nd Tble 1, re quite striking. The three inititor trnas, which differ considerbly in their nucleotide sequence, nonetheless hve common pttern of digestion by the SI endonuclese, showing two susceptible sites in the nticodon loop. In contrst, the chin-elongting trnas, including two of the internl methionine trnas, show mrkedly different pttern of Si nuclese susceptibility in the nticddon loop. Except for nucleotides tht re resistnt to SI digestion becuse of modifiction, the digestion ptterns of ll the chin-elongting trnas re similr to ech other with four susceptible sites, one of which, U33, my be secondry site. These results suggest tht the conformtion of the nticodon loop is similr in ll inititor trnas nd tht this conformtion differs from tht seen in elongtion trnas. As judged by this criterion, there ppers to be substntil similrity in the conformtion of the nticodon loop of ll chin-elongting trnas. It is interesting tht the results given by this enymtic structurl probe differ considerbly from erlier ttempts to find inititor-elongtion trna differences. For exmple, there hve been number of chemicl modifiction studies of both inititor nd chin-elongting trnas (for review, see refs. 13 nd 14) whose results re quite complex, but they cnnot be interpreted to indicte conformtionl differences. Likewise, oligonucleotide binding studies (15) of these trna molecules do not show significnt differences. Thus, the nture of the conformtionl chnge tht we hve detected is likely to be subtle nd not redily observble by these other techniques. In the present cse we re using the structurl specificity of n endonuclese to detect t4e conformtionl difference. We must emphsie tht our results re bsed on kinetic studies using Si nuclese, nd tht the differences we observe re differences in initil clevge sites. Prolonged digestion of the inititor trnas might show other sites susceptible to Si nuclese. However, our interest lies only with the identity of initil clevge sites since these reflect the conformtion of the intct trna. S1 nuclese clevge sites in nticodon loop of vrious trnas Residue no Anticodon Inititor trnas E. coli trnafmet Cm U C; Al U A A Yest trnafmet C U Cl Al U t6a A Mmmlin trnafmet C C C; Al U t6a A Elongtion trnas E. coli trnammet C Ul c4c Al Ul t6a A Yest trnammet C Ul Cl Al Ul t6a A Yest trnaphe Cm Ul Gm Al Al Y A Schio. pombe trnaphe Cm Ul Gm Al Al Y A E. coli trna2glu C Ul Ml Ul C m2a C E. coli trnatrp (CA 16) Cm Ul Cl Cl Al ms2i6a A S1 nuclese clevge sites re indicted by I fter the bse. M is 2-thio-5-methylminomethyl uridine. Y is wybutosine. Other bbrevitions re stndrd (1).

5 Biochemistry: Wrede et l. Wht then provides the physicl bsis for these differences in nucleotide sensitivity? It is cler tht the sequence of nucleotides in the nticodon loops cnnot be responsible for this difference since the nticodon loop sequence of yest trnafmet nd yest trnammet re identicl (Fig. 1). However, the nticodon stems do provide suggestion regrding the source of the differences. As shown in Fig. 1 A-C, the three bse pirs in the nticodon stem proximl to the loop in inititor trnas re ll the sme: there re three gunosine residues on the 5' side nd three cytosine residues on the 3' side. No chin-elongting trnas hve this pttern of bse piring in the nticodon stem (1). Furthermore, this is the only feture common to inititor trna molecules. As discussed bove, there re significnt differences in the T4C loop, in the hydrogen bonding of the cceptor stem, nd in the nture of the pyrimidine in position 33 of inititor trnas. The conformtion of the loop my influenced by the type of bse pirs tht re found in the djoining stem. Preliminry experiments tht we hve crried out with frgmented inititor trna molecules from yest pper to support this suggestion. The trna frgments re mde by cleving the molecule in the T-loop nd removing the 3' frgment. The nuclese susceptibility of the remining threequrter molecule is identicl to tht of the intct molecule. This is consistent with the ide tht the nticodon stem fixes the conformtion of the nticodon loop. The influence of stem sequences on the loop conformtion is further supported by nuclese digestion experiments on Schio. pombe trnaphe (to be reported elsewhere). This RNA hs the sme D-loop s yest trnaphe, but the D stem sequence is different. The susceptibility of Schio. pombe trnaphe in the D-loop to TI nuclese differs mrkedly from tht of yest trnaphe. Our results with the three-qurter frgment yest trnafmet re consistent with those reported by Dube et l. (16). They found tht frgments of E. coli inititor trna could bind to the ribosoml P site in the presence of the codon AUG. Among the frgments tht could bind were those contining just the nticodon loop nd prt of the nticodon stem. Specificlly, frgments contining t lest two of the GC pirs in the nticodon stem suffice to fix n nticodon loop conformtion tht is recognied s belonging to n inititor trna. An interesting cse tht should be exmined further is the inititor trna from the mitochondri of Neurospor crss. Unlike the other inititors discussed here, this trna shows only two GC bse pirs in the nticondon stem. It will be interesting to see if this inititor hs the nticodon loop conformtion reported here, prticulrly in light of the fct tht it is known to function s n inititor in n in intro E. coli protein synthetic system (17). It is possible tht in trnas with only two nticodon stem GC bse pirs djcent to the loop, the nticodon loop my ssume two conformtions: tht chrcteristic of elongtion trnas nd tht chrcteristic of inititors. In this regrd, it will be useful to exmine the nuclese digestion pttern of E. coli or yest trnaarg nd yest trnalys, ll of which hve two GC pirs proximl to the loop. These studies indicte tht there is conformtion of the nticodon loop tht is unique to inititor trna molecules. By impliction, this is likely to be n importnt considertion in trgeting the inititor for the ribosoml P site. Although we hve no evidence of the nture of the conformtionl difference from the present study, the fct tht chemicl modifiction nd oligonucleotide binding studies do not show significnt differences between inititors nd elongtion trnas suggests tht the Proc. Nti. Acd. Sci. USA 76 (1979) 3293 conformtionl distinction my be smll in mgnitude. Accordingly, one my sk whether it is possible tht trna molecules, in going from the A site to the P site of ribosome, undergo this conformtionl shift. Is it possible tht in the ribosoml P site ll trna molecules hve n nticodon loop conformtion similr to tht seen here for the inititor trnas? We suggest tht the energy brrier between these different conformtions is not gret nd tht the shift could be ssocited with trnsloction inside the ribosome. Experiments cn be crried out to test this hypothesis directly. In prticulr, it my be possible to chnge the environment of chin-elongting trnas in such wy s to modify the conformtion of the nticodon loop so tht the chnge cn be detected by nuclese structurl probes. It would be of gret interest to exmine trnas such s E. coli nd yest trnaarg or yest trnalys, which my be ble to shift conformtions more redily s result of the two GC pirs in their nticodon stems. Inititor nd chin-elongtion trna molecules pper to be subtle structures tht re grossly similr but nonetheless hve distinct nticodon loop conformtions with importnt functionl consequences. It will be of gret interest to define the physicl nture of the conformtion differences between these two clsses of trna molecules. We thnk Drs. B. A. Roe, L. H. Schulmn, U. L. RjBhndry, H. Feldmnn, D. S611, H. Donis-Keller, J. Vournkis, nd R. Buckinghm for their generous help in supplying mterils. We cknowledge the technicl ssistnce of Lorrin Yee nd J. Simpson for ssistnce in prepring the mnuscript. This reserch ws supported by grnts from the Ntionl Institutes of Helth, the Ntionl Science Foundtion, the Ntionl Aeronutics nd Spce Administrtion, nd the Americn Cncer Society. P.W. is recipient of fellowship from the Deutsche Forschungsgemeinschft. 1. Guss, D. H., Gruter, F. & Sprinl, M. (1979) Nucleic Acids Res. 6, rl-r Dube, S. K. & Mrcker, K. A. (1969) Eur. J. Biochem. 8, Simsek, M. & RjBhndry, U. L. (1972) Biochem. Biophys. Res. Commun. 49, Simsek, M., RjBhndry, U. L., Boisnrd, M. & Petrissnt, G. (1974) Nture (London) 247, RjBhndry, U. L. & Ghosh, H. P. (1969) J. Biol. Chem. 244, Lengyel, P. (1974) in Ribosomes, Cold Spring Hrbor Monogrph Series, eds. Nomur, M., Tissieres, A. & Lengyel, P. (Cold Spring Hrbor Lbortory, Cold Spring Hrbor, NY), pp Hrd, F. & Dhlberg, J. B. (1975) Nucleic Acids Res. 2, Wurst, R., Vournkis, J. & Mxm, A. (1978) Biochemistry 17, Wrede, P., Wurst, R., Vournkis, J. & Rich, A. (1979) J. Biol. Chem., in press. 1. Khn, M. S. N. & Mden, B. E. H. (1976) FEBS Lett. 72, Dodgson, J. B. & Wells, R. D. (1977) Biochemistry 16, Donis-Keller, H., Mxm, A. & Gilbert, W. (1977) Nucleic Acids Res. 4, Rich, A. & RjBhndry, U. L. (1976) Annu. Rev. Biochem. 45, Goddrd, J. P. (1977) Prog. Biophys. Mol. Biol. 32, Uhlenbeck,. C. (1972) J. Mol. Biol. 65, Dube, S. K., Rudlnd, P. S., Clrk, B. F. C. & Mrcker, K. A. (1969) Cold Spring Hrbor Symp. Qunt. Biol. 34, Heckmn, J. E., Hecker, L. I., Schwrtbch, S. D., Brnett, W. E., Bumstrk, B. & RjBhndry, U. L. (1978) Cell 13,