The fi del ity of the trans la tion of the ge netic code *.

Transcription

1 Vol. 48 No. 2/ QUARTERLY Review The fi del ity of the trans la tion of the ge netic code *. Rajan Sankaranarayanan and Dino Moras Laboratoire de Biologie et Genomique Structurales, IGBMC, CNRS/INSERM/ULP, BP Illkirch Cedex, Strasbourg, France Received: 14 February, 2001 Key words: ge netic code, trans la tion, aminoacyl-trna synthetase, ed it ing, evo lu tion Aminoacyl-tRNA syn the tas es play a cen tral role in main tain ing ac cu racy dur ing the trans la tion of the ge netic code. To achieve this chal leng ing task they have to dis crim i - nate against amino ac ids that are very closely re lated not only in struc ture but also in chem i cal na ture. A dou ble-sieve ed it ing model was pro posed in the late sev en ties to ex plain how two closely re lated amino ac ids may be dis crim i nated. How ever, a clear un der stand ing of this mech a nism re quired struc tural in for ma tion on syn the tas es that are faced with such a prob lem of amino acid dis crim i na tion. The first struc tural ba sis for the ed it ing model came re cently from the crys tal struc ture of isoleucyl-trna synthetase, a class I synthetase, which has to dis crim i nate against valine. The struc - ture showed the pres ence of two cat a lytic sites in the same en zyme, one for ac ti va tion, a coarse sieve which binds both isoleucine and valine, and an other for ed it ing, a fine sieve which binds only valine and re jects isoleucine. An other struc ture of the en zyme in com plex with trna showed that the trna is re spon si ble for the translocation of the misactivated amino-acid sub strate from the cat a lytic site to the ed it ing site. These stud ies were mainly fo cused on class I syn the tas es and the sit u a tion was not clear about how class II en zymes dis crim i nate against sim i lar amino ac ids. The re cent struc tural and en zy matic stud ies on threonyl-trna synthetase, a class II en zyme, re - veal how this chal leng ing task is achieved by us ing a unique zinc ion in the ac tive site as well as by em ploy ing a sep a rate do main for spe cific ed it ing ac tiv ity. These stud ies Pres ented at the In ter na tional Con fer ence on Mo lec u lar Ar chi tec ture of Evo lu tion, Pri mary and Sec - ondary Determinants Poznañ, Poland, Oc to ber 29 31, The work was supported by grants from EU project No. BIO , CNRS, INSERM, ULP and Ministére de la Re cher che et de la Technologie. Corresponding author: moras@igbmc.u-strasbg.fr; sankar@igbmc.u-strasbg.fr; fax: Abbreviation: aars, aminoacyl-trna synthetase.

2 324 The fi del ity of the trans la tion of the ge netic code 2001 led us to pro pose a model which em pha sizes the mir ror sym met ri cal ap proach of the two classes of en zymes and high lights that trna is the key player in the evo lu tion of these class of en zymes. A high fi del ity is re quired in re ac tion mech a - nisms that are in volved in the information transfer from the ge netic code. Dur ing trans - la tion of the ge netic mes sage, amino - acyl-trna synthetases (aarss) play a very crucial role in main tain ing such a high ac cu - racy (Carter, 1993; Schimmel & Söll, 1979). To achieve this chal leng ing task they have to spe cif i cally rec og nize one amino acid out of a pool of twenty pro tein amino ac ids and a few non protein amino ac ids, as well as one cog - nate trna fam ily out of twenty trna fam i lies (Jakubowski & Goldman, 1992). Spe cific rec - ognition of trna molecules is not a ma jor problem since they are large enough to pro - vide a wide range of spe cific in ter ac tions with the cor re spond ing synthetase (Giege et al., 1998). It was shown that the error rate in trna selection is of the order of 10 6 or lower. How ever, the sit u a tion with the amino-acid sub strates is very dif fer ent in that they are much smaller and there are a few amino ac ids which have very similar, either struc tur ally or chem i cally, side chains. In fact, such a problem of amino acid discrimination was first posed by Linus Pauling in 1957 (Pauling, 1957). Ac cord ing to him, if there is only a dif fer ence of a sin gle methyl group be - tween two sub strates, the dif fer ence in their binding energies would not al low the er ror rate to be better than 1 in 5. How ever, the ob - served er ror rates in the se lec tion of amino ac - ids by their cognate synthetases are much lower, in the range of 10 4 to 10 5 (Loftfield & Vanderjagt, 1972). Numerous studies were made over the years to understand how syn - the tas es over come the prob lem of amino acid discrimination. A clear structural ba sis on how these en zymes achieve such a high ac cu - racy in se lect ing their cog nate amino-acid sub - strates is just be gin ning to emerge. This in - volves a process called editing or proof-read - ing by syn the tas es that are faced with such a problem (Fersht, 1985). This re view fo cuses on the dra matic leap in our un der stand ing of how synthetases discriminate against very closely related amino-acid sub strates, made pos si ble by the re cent dis cov ery of sev eral key crystal struc tures. THE SYNTHETASE FAMILY AaRSs perform the task of at tach ing an amino acid to the ter mi nal ribose of their cog - nate trna mol e cules through a two-step re ac - tion called aminoacylation re ac tion. In the first step, the amino acid is com bined with an ATP mol e cule to form an aminoacyl adenylate intermediate. In the sec ond step, the amino acid moiety is transferred to the trna mole - cule. These en zymes were broadly par ti tioned into two classes of 10 en zymes each, as a re - sult of the surprising discovery of the exis - tence of two fundamentally dif fer ent active sites (Ta ble 1) (Eriani et al., 1990). The class I enzymes have a Rossmann-fold (Rossmann et al., 1974) cat a lytic do main whereas in class II enzymes it is based on an antiparallel β-fold flanked by α-he li ces (Cusack et al., 1990; Rould et al., 1989; Ruff et al., 1991). With a few ex cep tions, the par ti tion ing is highly cor - re lated with struc tural and func tional char ac - ter is tics (Arnez & Moras, 1997; Cusack, 1995; Moras, 1992). Class I enzymes are gen er ally mono mers whereas the class II en zymes ex ist as dimers. The class I en zymes ap proach their trna sub strates from the mi nor groove of the ac cep tor stem and attach the amino acids to the 2 OH group of the ter mi nal ribose. They possess two sig na ture motifs HIGH and KMSKS which are re spon si ble for sub strate rec og ni tion. In the class II fam ily, the trna is ap proached from the ma jor groove side of the ac cep tor stem and aminoacylated at the 3 OH group, with the exception of PheRS. Class II enzymes pos sess three highly conserved mo - tifs. Mo tif 1 is mainly responsible for the

3 Vol. 48 R. Sankaranarayanan and D. Moras 325 dimerization of the sub units, whereas the other two mo tifs pos sess some key con served res i dues which are in volved in po si tion ing of the sub strates and in the re ac tion mech a nism. Apart from these class-specific fea tures, the en zymes within one class could be grouped de - pend ing on sim i lar i ties ex hib ited among them. This group ing could de pend ei ther upon the pos ses sion of sim i lar mod ules, since aarss are mod u lar pro teins (Delarue & Moras, 1993; Sankaranarayanan & Moras, THE PROBLEM OF AMINO ACID DISCRIMINATION The ac cu racy of the aminoacylation re ac tion de pends on the abil ity of aarss to spe cif i cally rec og nize the cor rect amino acid and to at tach it to the cognate-trna. The trna molecules are large enough to pres ent the aarss with a large sur face area for in ter ac tion. This fa cil i - tates the se lec tion pro cess and thus the er ror in trna selection is in the range of 10 6 or Ta ble 1. Clas si fi ca tion of aminoacyl-trna syn the tas es on the ba sis of struc tural and func tional or ga - ni za tion. The en zymes for which the struc ture in the apo form or in com plex with the trna is known are un der lined or given in bold, re spec tively. The en zymes which are shown to pos sess an ed it ing ac tiv ity are in di cated by an as - ter isk. Group a Class I Qua ter nary struc ture Class II Qua ter nary struc ture CysRS α GlyRS α 2 ValRS* α AlaRS* α, α 4 LeuRS* α, α 2 SerRS α 2 IleRS * α ProRS* α 2 MetRS* α 2 ThrRS* α 2 Group b ArgRS α HisRS α 2 GlnRS α AspRS α 2 GluRS α, αβ AsnRS α 2 Group c LysRS α LysRS α 2 TyrRS α 2 PheRS* α 2 β 2 TrpRS α 2 GlyRS α 2 β ), or upon higher con ser va tion in their se - quence. Based on these considerations, the two classes are di vided into three subgroups each. More over, idiosyncratic features that could cor re spond to spe cific func tions are also seen in aarss per tain ing to each of the twenty amino acids or even in the same system but from di verse spe cies. lower. As seen from sev eral crys tal struc tures of aars trna com plexes, the synthetases have evolved dif fer ent domains that spe cif i - cally rec og nize the anticodon arm (Arnez et al., 1995; Cavarelli et al., 1993; Cusack et al., 1998; Logan et al., 1995; Rould et al., 1991; Ruff et al., 1991; Sankaranarayanan et al., 1999), the acceptor arm (Sankaranarayanan

4 326 The fi del ity of the trans la tion of the ge netic code 2001 et al., 1999) and in some specific cases the variable loop (Biou et al., 1994) of the trna mol e cule, to achieve a high spec i fic ity in trna recognition. In con trast, the amino acids are much smaller molecules and therefore their recognition poses a more difficult problem. For most of the amino acids, the side chains ex hibit strong and unique chem i cal char ac ter - is tics so that their se lec tion does not pres ent a ma jor prob lem. How ever, side chains of some amino acids are struc tur ally or chemically quite sim i lar, and there fore their spe cific rec - ognition could be a ma jor prob lem, for ex am - ple in the case of valine, isoleucine, threonine, serine, alanine and glycine. This is typically the problem pointed out by Pauling (1957). How ever, his es ti ma tion of the er ror rate of 1 in 5 between two sub strates that dif fer by a sin gle meth y lene group is based on a dif fer - ence in bind ing en ergy of 1 kcal/mol. This en - ergy dif fer ence cor re sponds to a meth y lene group trans ferred from a hy dro philic to a hy - dro pho bic sol vent. Later, this value was es ti - mated to be much higher, 3.4 kcal/mol per methylene group, as the bind ing is more spe - cific in pro teins than in hy dro pho bic sol vents (Fersht et al., 1980). There fore, the er ror rate cor re spond ing to such a dif fer ence in bind ing is 1 in 200. Even this rate is much higher than what is ob served ex per i men tally in the case of syn the tas es where the er ror is in the range of 10 4 to 10 5 (Loftfield & Vanderjagt, 1972). There fore, the question re mained as to how do aarss achieve such a high spec i fic ity in dis - criminating against sim i lar sub strates?. EDITING OR PROOFREADING ACTIVITY The so lu tion to the problem of amino acid discrimination came mainly from the stud ies on the en zymes IleRS and ValRS. An editing activity was first dis cov ered in IleRS, which spe cif i cally hy dro lyzed the in cor rectly formed products, either valyl adenyalte (Baldwin & Berg, 1966) or val-trna Ile (Eldred & Schim - mel, 1972). Since it is not pos si ble for a pocket designed for binding isoleucine to dis crim i - nate com pletely against valine, which is smaller by only a methylene group, valine is ac ti vated and charged on the trna with a fre - quency of approximately 1 in 300. However, the over all er ror rate in pro tein biosynthesis, i.e. the misin corporation of valine in stead of isoleucine in a grow ing polypeptide chain, is only 1 in This higher rate is achieved through the ed it ing re ac tion, which en hances the dis crim i na tion ra tio by a fac tor of 10. The ed it ing reaction in this enzyme can oc cur through a pre-transfer or a post-transfer path way. The pre-transfer mech a nism in - volves the hy dro ly sis of the in cor rectly formed valyl adenylate (Baldwin & Berg, 1966) whereas in the post-transfer mech a - nism the enzyme deacylates the in cor rectly charged val-trna Ile (Eldred & Schimmel, 1972). A sim i lar rec og ni tion problem is also faced by ValRS which has to discriminate against the isosteric threonine. This enzyme also was shown to pos sesses an ed it ing ac tiv - ity to elim i nate the in cor rectly formed threonyl adenylate or thr-trna Val (Fersht & Kaethner, 1976). Based on these re sults a dou - ble-sieve ed it ing model for the se lec tion of sim i lar sub strates was pro posed by Alan Fersht (Fersht, 1985; Fersht & Dingwall, 1979). Ac cord ing to this model, the enzyme first binds amino acids that are sim i lar or smaller than the cor rect sub strate and re jects the ones that are big ger, us ing a first site named the coarse-sieve. Then, in a sec ond step, the smaller sub strates are se lec tively bound and hy dro lyzed us ing a fine-sieve. Even though the el e gant dou ble-sieve ed it ing model could well explain the editing mechanism in IleRS and ValRS, it could not com pletely ex - plain the ed it ing ac tiv ity found in a few other synthetases, for ex am ple MetRS, PheRS and AlaRS. Dif fer ent path ways were pro posed to account for these cases, which will be dis - cussed briefly later.

5 Vol. 48 R. Sankaranarayanan and D. Moras 327 AMINO ACID RECOGNITION BY THE CLASS I IleRS Most of our current understanding on the ed it ing ac tiv ity co mes from the bio chem i cal and struc tural studies on IleRS. Par tic u larly in the last few years, there has been a surge of information on the ed it ing ac tiv ity of this en - zyme resulting from the dis cov er ies of the crys tal struc tures of the en zyme in com plexes with different sub strates (Nureki et al., 1998) as well as with the trna (Silvian et al., 1999). IleRS is a class I enzyme and be longs to the subgroup Ia along with enzymes specific for valine, cysteine, leucine, arginine and methionine. Its active site is based on a Rossmann-fold do main con sist ing of al ter nat - ing β-strands and α-he li ces form ing a β 6 α 4 structure. A spe cial char ac ter is tic of IleRSs from dif fer ent or gan isms is that they all pos - sess an in ser tion in the Rossmann-fold cat a - lytic domain of ap prox i mately 200 residues which is called the con nec tive polypeptide 1 (CP1) (Starzyk et al., 1987). Mutational stud - ies in a seg ment of the CP1 frag ment have shown that it al ters the hy dro ly sis of val-trna Ile, dem on strat ing that it con tains a catalytic center for the ed it ing re ac tion. Fur - thermore, the CP1 frag ment alone expressed as an in de pend ent protein could also spe cif i - cally hy dro lyze the incorrect product (Lin et al., 1996). However, the first structural ev i - dence of the double-sieve model came from the crystal structure of IleRS (Fig. 1A) from Thermus thermophilus solved in com plex with the amino-acid sub strates isoleucine or valine (Nureki et al., 1998). The structure of the en - zyme with isoleucine showed its pres ence only in the Rossmann-fold catalytic do main. How - ever, in the crys tals soaked with valine, the amino acid bound to both the cat a lytic do main and to a site in the CP1 fragment. The struc - ture showed clearly that the activation site (coarse-sieve) could bind both isoleucine and Fig ure 1. The struc ture of IleRS com plexes. A) Mod u lar struc ture of IleRS from Thermus thermophilus (Nureki et al., 1998). The cat a lytic do main is in di cated in green, the ed it ing do main in pink, the anticodon bind ing mod ule in or ange and the co or di na tion of zinc ion in yel - low. B) The struc ture of IleRS from Staph y lo coc cus aureus complexed with trna Ile (Silvian et al., 1999). The pro tein mod ules are in di cated as in Fig. 1A and the trna and the in hib i tor mol e cule mupirocin are in di cated in red and blue, re spec tively. All the fig ures ex cept Figs. 3B and 4 were drawn us ing SETOR (Ev ans, 1993).

6 328 The fi del ity of the trans la tion of the ge netic code 2001 valine, whereas larger amino ac ids, in clud ing leucine, are rejected by the enzyme due to steric hin drance. In the CP1 mod ule, only valine can bind and isoleucine can not fit in the ed it ing pocket (fine-sieve) be cause of steric hindrance with res i dues lin ing the pocket. This study, there fore, is a clear dem on stra tion of the double-sieve mech a nism of editing. However, it was not clear as to how the sub - strate (ei ther val-amp or val-trna Ile ) translocation oc curs from the cat a lytic site to the ed it ing site which is more than 25 Å away. The struc ture of IleRS from Staphylococcus aureus complexed with trna Ile and mupirocin (Fig. 1B) suggested a par tial an - swer to the translocation problem (Silvian et al., 1999). In this structure, the class I con - served Rossmann-fold ac tive site mod ule con - tained the mupirocin molecule. In an earlier study, it has been shown that three nu cleo - tides in the D loop of the trna are es sen tial for trna de pend ent editing activity (Hale et al., 1997). In the structure, the enzyme does not in ter act with the D loop and there fore it is not clear how the D loop can in flu ence the ed - it ing ac tiv ity. In ter est ingly, the trna Ile ac cep - tor stem, even though only vis i ble till Cy to sine 74, is in a he li cal form which is typ i cal of free trna (Robertus et al., 1974; Suddath et al., 1974) or trnas bound to the active site of class II en zymes (Biou et al., 1994; Ruff et al., 1991). If the he li cal con for ma tion of the ac cep - tor end is mod eled be yond cy to sine 74, the 3 end of the trna can not reach the ac tive site in the Rossmann-fold do main. How ever, the ter - minal adenosine is point ing to wards the CP1 frag ment, interacting par tic u larly with res i - dues His 392 and Tyr 394 which have been shown to be di rectly in volved in the ed it ing ac - tiv ity (Schmidt & Schimmel, 1995). Thus, the struc ture prob a bly rep re sents an editing complex. The study showed that the trna mol e cule is di rectly in volved in the translocation event, wherein the CCA-end shut tles from the ac tive site to the ed it ing site by shift ing from a hair pin con for ma tion to he - li cal con for ma tion. Therefore, the model for post-transfer ed it ing is very sim i lar to that ob - served in the case of DNA poly mer ase I where the na scent strand shut tles be tween the ac tive site and the ed it ing site (Brutlag & Kornberg, 1972; Freemont et al., 1988; Joyce & Steitz, 1994). A similar shuttling of the noncognate valyl adenylate has also been shown to be re - spon si ble for pre-transfer ed it ing by a ki netic study with a flu o res cent probe (Nomanbhoy et al., 1999). The mechanism of ed it ing could also be very similar in a related class I en - zyme, ValRS, which has to dis crim i nate against threonine. In deed it ac ti vates threonine at an error rate of 1 in 350 to 1 in 400 (Lin & Schimmel, 1996). The en zyme also possesses the CP1 frag ment re spon si ble for hydrolyzing the incorrect thr-trna Val (Lin et al., 1996). AMINO ACID RECOGNITION BY THE CLASS II ThrRS Even though a large amount of bio chem i cal and struc tural data were avail able on the ed it - ing ac tiv ity of syn the tas es, most of them were dedicated to class I en zymes. Very lit tle is known about how class II enzymes dis crim i - nate against closely re lated amino acids. In fact, the can di dates for ed it ing in class II are not many. The two en zymes that typ i cally fit into the cat e gory that require editing are AlaRS and ThrRS. AlaRS has to mainly dis - criminate against glycine, which is shorter than alanine by one meth y lene group. ThrRS faces a com plex dis crim i na tion prob lem against two closely re lated amino ac ids. It has to dis crim i nate against valine, which is isosteric with threonine, and serine, which is shorter by one methyl group. ThrRS is a class II enzyme which is based on an antiparallel β-fold for the cat a lytic mod ule (Arnez & Moras, 1997). The struc ture of ThrRS from Escherichia coli in complex with its trna showed that the en zyme is a mod u lar pro tein made of four do mains (Fig. 2A) (Sankara- narayanan et al., 1999). The N-terminal mod -

7 Vol. 48 R. Sankaranarayanan and D. Moras 329 ules char ac ter is tic of the threonyl sys tem fold into two sep a rate do mains and are con nected to the cat a lytic mod ule through a linker he lix. Surprisingly, the structure showed for the first time the pres ence of a zinc ion in the ac - tive site mod ule of an aars, at a po si tion close to the amino acid bind ing pocket. Struc tural zinc ions have been found in other syn the tas - es, for ex am ple in MetRS (Brunie et al., 1990) and IleRS (Nureki et al., 1998), but not in the active site. The zinc ion is co or di nated by three pro tein lig ands and by a wa ter mol e cule. The strict con ser va tion of the zinc bind ing res - idues throughout evolution and mutational stud ies in vivo showed that the zinc ion has a cru cial role to play in amino acid rec og ni tion. Also, the co or di na tion of the zinc ion by a wa - class II synthetases, thus ob vi at ing the need for a struc tural metal cat ion to stabilize the fold (Arnez & Moras, 1997). Two crys tal struc tures of a trun cated ver sion of the en zyme (con sist ing of the cat a lytic and anticodon bind ing domains) complexed with threonine or a threonyl-adenylate an a log clearly showed a di rect role for the zinc ion in amino acid rec og ni tion (Sankaranarayanan et al., 2000). Upon threonine bind ing, the wa ter molecule is replaced by the threonine sub - strate and the zinc ion in ter acts with both the amino group and the side-chain hydroxyl of the sub strate (Fig. 2B). The zinc ion changes its co or di na tion from tetrahedral, in the ab - sence of the sub strate, to square-based py ram - i dal in the pres ence of the sub strate. Thus, the Fig ure 2. The struc ture of ThrRS from Esch e richia coli. A) Do main ar chi tec ture of a mono mer ThrRS. Dif fer ent col ors are used to in di cated the dif fer ent mod ules of the en - zyme. B) The ac tive site of the trun cated form of ThrRS show ing the in ter ac tion of the zinc ion with the amino-acid sub strate. The con served mo tifs 2 and 3 are in di cated in red and green, re spec tively. The zinc ion (pink) and a wa ter mol e cule (cyan) are rep re sented as spheres. ter mol e cule sug gested that it may have a cat a - lytic role (Christianson, 1991). A purely struc - tural role for the zinc ion could be ruled out since it is found within the active site and is coordinated to a wa ter molecule. More over, the class II catalytic do main has a sta ble ter - tiary fold as found in the struc tures of other zinc ion ful fils a new func tion which is nei ther cat a lytic nor struc tural, but acts as a co fac tor in the amino acid rec og ni tion process. This mode of in ter ac tion of threonine with the zinc ion clearly showed that the isosteric valine would be re jected by the en zyme, since one of the side chain methyl groups would be in an

8 330 The fi del ity of the trans la tion of the ge netic code 2001 un fa vor able con tact with the zinc ion. Amino acid ac ti va tion ex per i ments showed that valine is in deed com pletely re jected by the en - zyme. How ever, the stud ies showed that serine is activated by the enzyme with a 1000-fold re duced ef fi ciency. Even though this er ror rate is slightly lower than that ob - served for the re jec tion of valine by IleRS and threonine by ValRS at the activation step (Jakubowski & Goldman, 1992), it is higher than that ob served in vivo. There fore, an ed it - ing mechanism it is nec es sary to cor rect the er ror. A typ i cal char ac ter iza tion of the ex is tence of a pre-transfer ed it ing mech a nism in volves the mea sure of ATP hy dro ly sis in the pres ence of noncognate sub strates, as observed in the in the presence of a seryl adenylate analog showed that serine in ter acts with the zinc ion in a way that is very similar to threonine (Dock-Bregeon et al., 2000). This fur ther rules out the pos si bil ity that the zinc ion could have a cat a lytic role or that the metal ion could be re spon si ble for hy dro lyz ing the in cor rect adenylate, since such a mech a nism would re - quire a different mode of binding of serine compared to threonine. Enzymatic mea sure - ments carried out to find out whether a post-transfer ed it ing mechanism ex ists in ThrRS showed clearly that the enzyme uses its N-terminal mod ule for se lec tive hy dro ly sis of the in cor rectly formed Ser-tRNA Thr (Dock-Bregeon et al., 2000). A struc tural su - per po si tion (Fig. 3A) of the ac cep tor arm of Fig ure 3. The ed it ing site of ThrRS. A) Struc tural su per po si tion of the ac cep tor arm of trna Gln on the ThrRS trna Thr com plex struc ture, show ing the ac cep tor end of trna Gln point ing to wards a site in the N2 do main of ThrRS. B) Sur face rep re sen ta tion of the N2 do - main, where the ed it ing site is in di cated by an ar row. Fig ure drawn us ing GRASP (Nicholls& Honig, 1991). Adapted from Dock-Bregeon et al. (2000). case of some class I syn the tas es and in AlaRS. In the case of ThrRS, no ATP consumption could be de tected, in di cat ing that no er ror cor - rec tion takes place at the level of the adenylate. The crys tal struc ture of the en zyme trna Gln, from the class I GlnRS trna Gln com plex (Rould et al., 1989), on top of ThrRS (Sankaranarayanan et al., 1999) showed the CCA-end of trna Gln point ing to wards a pocket in the N2 module of ThrRS (Fig. 3B).

9 Vol. 48 R. Sankaranarayanan and D. Moras 331 This pocket has been im pli cated ear lier as re - sponsible for the editing activity of ThrRS (Sankaranarayanan et al., 1999). Mutational stud ies of the res i dues sur round ing this pocket have shown that it is indeed responsi - ble for the ed it ing activity (Dock-Bregeon et al., 2000). Thus, the conformation of the CCA-end of the trna changes from he li cal, as - sumed in or der to reach the ac tive site, to bent which is necessary to reach the ed it ing site. The incorrectly aminoacylated serine moi ety is translocated to the ed it ing site by the trna and gets hy dro lyzed. This mech a nism of ed it - ing to gether with the ac ti va tion mech a nism of a class II synthetase can be considered a mir - ror im age of the events in a class I synthetase (Fig. 4). In ThrRS, the over all mech a nism fol lows the gen eral dou ble-sieve model for ed it ing. How - ever, in con trast to what has been pro posed so far, the first is a chem i cal sieve and not a steric one. This was illustrated by the amino acids that pos sess a hydroxyl group attached to the β-po si tion. There fore, size is not a strict cri te rion at the coarse-sieve. Through a trna mediated post-transfer ed it ing ac tiv ity, the smaller amino acid is then selectively hy dro - lyzed us ing the sec ond fine-sieve. EDITING IN OTHER SYSTEMS In ad di tion to the pre- and post-transfer ed it - ing reactions de scribed before, a third path - way is used by MetRS to main tain the accu - racy of the aminoacylation re ac tion (Jaku- bowski & Fersht, 1981). In this case, the en - zyme misactivates homocysteine, which has been shown to cyclize to form homocysteine thiolactone. LeuRS from E. coli possesses post-transfer ed it ing ac tiv ity to wards two analogous non-protein amino acids gammahydroxyleucine and homocysteine (Englisch et al., 1986). The class II PheRS has also been Fig ure 4. A model of the ed it ing pro cess in both classes of syn the tas es em pha siz ing the sym met ri cal na - ture of the ac ti va tion and ed it ing mech a nism. Adapted from Dock-Bregeon et al. (2000). acid ac ti va tion ex per i ments with an un nat u ral amino acid β-hydroxynorvaline which is big - ger than threonine by one meth y lene group. This amino acid could be ac ti vated by ThrRS (Sankaranarayanan et al., 2000), show ing that the en zyme uses the zinc ion to select amino shown to spe cif i cally deacylate the mis - charged Ile-tRNA Phe (Yarus, 1972). AlaRS has been shown to pos sess an ed it ing ac tiv ity for both glycine and serine (Tsui & Fersht, 1981). In this case, glycine fits in the model of a dou - ble-sieve ed it ing and it re mains to be seen how

10 332 The fi del ity of the trans la tion of the ge netic code 2001 the ac tive site of AlaRS can ac ti vate serine. It is also in ter est ing to note that the N2 do main responsible for the editing of ser-trna Thr in ThrRS is also present in AlaRS (Sankara- narayanan et al., 1999). Both bio chem i cal and structural studies are required to find out whether this do main is in volved in the ed it ing activity of AlaRS and to elucidate the editing mech a nism. Very re cently, it has been shown that ProRS also pos sesses an ed it ing ac tiv ity to hy dro lyze the micharged ala-trna Pro (Beuning & Musier-Forsyth, 2000). The en - zyme ac ti vates alanine times less ef fi - ciently than the cognate sub strate proline. It is ar gued that since the in vivo con cen tra tion of alanine (148 µm) in E. coli cells is much higher than that of proline (9 µm) (Raunio & Rosenqvist, 1970), the enzyme needs to pos - sess an ed it ing activity to main tain the ac cu - racy in the translation of the genetic code. How ever, it re mains to be seen how these en - zymes edit the noncognate amino acids. CONCLUSIONS AaRSs are thought to be an an cient fam ily of en zymes which pro vide a cru cial link be tween the RNA world and pro teins. It is be lieved that the early syn the tas es were made of only the catalytic mod ule charg ing mini RNA he li ces based on an op er a tional RNA code, more an - cient than the pres ent anticodon-based ge - netic code (Schimmel et al., 1993). Also, the evo lu tion of the anticodon arm of trna led to the evo lu tion of anticodon bind ing do mains in syn the tas es. Thus, the RNA mol e cule is a key player in the evo lu tion of the pres ent day syn - the tas es. As the evo lu tion ary pres sure on syn - thetases in creased to pro vide a much higher accuracy, they ac quired ad di tional mod ules re spon si ble for the ed it ing ac tiv ity. How ever, the evolutionary process is very much dic - tated by the ability of the CCA-end of the trna molecule to switch between two dif fer - ent con for ma tions (i.e. between he li cal and bent). It is intriguing that even though both classes of syn the tas es diverged very early in evo lu tion, they have ar rived at sim i lar mech a - nis tic so lu tions to the prob lem of main tain ing fi del ity in the trans la tion of the ge netic code. We thank Dr. Ber nard Rees for a very care - ful reading of the manu script. REF ER ENCES Arnez, J.G., Har ris, D.C., Mitschler, A., Rees, B., Francklyn, C.S. & Moras, D. (1995) Crys tal structure of histidyl-trna synthetase from Escherichia coli complexed with histi dyl-ade - nylate. EMBO J. 14, Arnez, J.G. & Moras, D. (1997) Structural and func tional con sid er ations of the amino - acylation reaction. Trends Biochem. Sci. 22, Baldwin, A.N. & Berg, P. (1966) Trans fer ri bo nu - cleic acid-induced hy dro ly sis of valyladenylate bound to isoleucyl ri bo nu cleic acid synthetase. J. Biol. Chem. 241, Beuning, P.J. & Musier-Forsyth, K. (2000) Hydrolytic ed it ing by a class II amino - acyl-trna synthetase. Proc. Natl. Acad. Sci. U.S.A. 97, Biou, V., Yaremchuk, A., Tukalo, M. & Cusack, S. (1994) The 2.9 Å crys tal struc ture of T. thermophilus seryl-trna synthetase com - plexed with trna(ser). Science 263, Brunie, S., Zelwer, C. & Risler, J.-L. (1990) Crys - tal lo graphic study at 2.5 Å res o lu tion of the in - teraction of methionyl-trna synthetase from Escherichia coli with ATP. J. Mol. Biol. 216, Brutlag, D. & Kornberg, A. (1972) En zy matic syn - thesis of deoxyribonucleic acid. 36. A proof - reading function for the 3 leads to 5 exo - nuclease activity in deoxyribonucleic acid polymerases. J. Biol. Chem. 247, Carter, C.W., Jr. (1993) Cognition, mechanism, and evo lu tion ary re la tion ships in amino - acyl-trna synthetases. Annu. Rev. Biochem. 62,

11 Vol. 48 R. Sankaranarayanan and D. Moras 333 Cavarelli, J., Rees, B., Ruff, M., Thierry, J.C. & Moras, D. (1993) Yeast trna Asp recognition by its cog nate class II aminoacyl-trna synthetase. Nature 362, Christianson, D.W. (1991) Structural biology of zinc. Adv. Pro tein Chem. 42, Cusack, S. (1995) Eleven down and nine to go. Na - ture Struct. Biol. 2, Cusack, S., Berthet-Colominas, C., Härtlein, M., Nassar, N. & Leberman, R. (1990) A sec ond class of synthetase struc ture re vealed by X-ray anal y sis of Esch e richia coli seryl-trna synthetase. Nature 347, Cusack, S., Yaremchuk, A., Krikliviy, I. & Tukalo, M. (1998) trna(pro) anticodon rec og ni tion by Thermus thermophilus prolyl-trna syn - thetase. Structure 6, Delarue, M. & Moras, D. (1993) The aminoacyl-trna synthetase family: Modules at work. Bioessays 15, Dock-Bregeon, A.C., Sankaranarayanan, R., Romby, P., Caillet, J., Springer, M., Rees, B., Francklyn, C.S., Ehresmann, C. & Moras, D. (2000) Transfer RNA mediated editing in threonyl-trna synthetase: The class II solu - tion to the dou ble dis crim i na tion prob lem. Cell 103, Eldred, E.W. & Schimmel, P.R. (1972) Rapid deacylation by isoleucyl transfer ribonucleic acid synthetase of isoleucine-specific transfer ri bo nu cleic acid aminoacylated with valine. J. Biol. Chem. 247, Englisch, S., Englisch, U., von der Haar, F. & Cramer, F. (1986) The proof read ing of hydroxy an a logues of leucine and isoleucine by leucyl-trna synthetases from E. coli and yeast. Nu cleic Acids Res. 14, Eriani, G., Delarue, M., Poch, O., Gangloff, J. & Moras, D. (1990) Par ti tion of trna syn the tas - es into two classes based on mu tu ally ex clu sive sets of se quence mo tifs. Nature 347, Evans, S.V. (1993) SETOR: hard ware lighted three-dimensional solid model rep re sen ta - tions of macromolecules. J. Mol. Graph ics 11, Fersht, A. (1985) Enzyme, Structure and Mech a - nism. Free man, San Fran cisco, New York. Fersht, A.R. & Dingwall, C. (1979) Evidence for the dou ble-sieve ed it ing mech a nism in pro tein synthesis. Steric exclusion of isoleucine by valyl-trna syn the tas es. Bio chem is try 18, Fersht, A.R. & Kaethner, M.M. (1976) En zyme hy - persensitivity. Rejection of threonine by the valyl-trna synthetase by misacylation and hydrolytic editing. Biochemistry 15, Fersht, A.R., Shindler, J.S. & Tsui, W.C. (1980) Probing the limits of pro tein-amino acid side chain recognition with the aminoacyl-trna synthetases. Discrimination against phenyl - alanine by tyrosyl-trna syn the tas es. Bio chem - is try 19, Freemont, P.S., Fried man, J.M., Beese, L.S., San - derson, M.R. & Steitz, T.A. (1988) Cocrystal structure of an editing complex of Klenow fragment with DNA. Proc. Natl. Acad. Sci. U.S.A. 85, Giege, R., Sissler, M. & Florentz, C. (1998) Uni ver - sal rules and idiosyncratic features in trna iden tity. Nu cleic Acids Res. 26, Hale, S.P., Auld, D.S., Schmidt, E. & Schimmel, P. (1997) Dis crete de ter mi nants in trans fer RNA for editing and aminoacylation. Science 276, Jakubowski, H. & Fersht, A.R. (1981) Alternative path ways for ed it ing non-cognate amino ac ids by aminoacyl-trna syn the tas es. Nu cleic Acids Res. 9, Jakubowski, H. & Goldman, E. (1992) Editing of er rors in se lec tion of amino ac ids for pro tein synthesis. Microbiol. Rev. 56, Joyce, C.M. & Steitz, T.A. (1994) Function and structure relationships in DNA polymerases. Annu. Rev. Biochem. 63, Lin, L., Hale, S.P. & Schimmel, P. (1996) Aminoacylation error correction [let ter]. Na - ture 384, Lin, L. & Schimmel, P. (1996) Mutational anal y sis sug gests the same de sign for ed it ing ac tiv i ties

12 334 The fi del ity of the trans la tion of the ge netic code 2001 of two trna syn the tas es. Biochemistry 35, Loftfield, R.B. & Vanderjagt, M.A. (1972) The fre - quency of errors in pro tein biosynthesis. Biochem. J. 128, Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) Crys tal structure of glycyl-trna synthetase from Thermus thermophilus. EMBO J. 14, Moras, D. (1992) Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 2, Nicholls, A. & Honig, B. (1991) A rapid fi nite dif - ference al go rithm uti liz ing suc ces sive over-re - lax ation to solve the Pois son-boltzmann equa - tion. J. Comp. Chem. 12, Nomanbhoy, T.K., Hendrickson, T.L. & Schimmel, P. (1999) Trans fer RNA-dependent trans - location of misactivated amino acids to pre - vent errors in pro tein synthesis. Mol. Cell 4, Nureki, O., Vassylev, D.G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T.L., Schimmel, P. & Yokoyama, S. (1998) En zyme struc ture with two cat a lytic sites for double-sieve selection of substrate. Science 280, Pauling, L. (1957) The prob a bil ity of er rors in the process of synthesis of pro tein molecules; in Festschrift für Pr. Dr. Ar thur Stoll, pp , Birkhauser Verlag, Basel. Raunio, R. & Rosenqvist, H. (1970) Amino acid pool of Escherichia coli during the different phases of growth. Acta Chem. Scand. 24, Robertus, J.D., Ladner, J.E., Finch, J.T., Rhodes, D., Brown, R.S., Clark, B.F.C. & Klug, A. (1974) Struc ture of yeast phenylalanine trna at 3 Å res o lu tion. Nature 250, Rossmann, M.G., Moras, D. & Olsen, K.W. (1974) Chem i cal and bi o log i cal evo lu tion of a nu cle o - tide-binding pro tein. Nature 250, Rould, M.A., Perona, J.J., Söll, D. & Steitz, T.A. (1989) Structure of E. coli glutaminyl-trna synthetase complexed with trna Gln and ATP at 2.8 Å res o lu tion. Science 246, Rould, M.A., Perona, J.J. & Steitz, T.A. (1991) Struc tural ba sis of anticodon loop rec og ni tion by glutaminyl-trna synthetase. Nature 352, Ruff, M., Krishnaswamy, S., Boeglin, M., Poter - szman, A., Mitschler, A., Podjarny, A., Rees, B., Thierry, J.C. & Moras, D. (1991) Class II aminoacyl trans fer RNA syn the tas es: Crys tal structure of yeast aspartyl-trna synthetase complexed with trna Asp. Sci ence 252, Sankaranarayanan, R., Dock-Bregeon, A.C., Rees, B., Bovee, M., Caillet, J., Romby, P., Fran - cklyn, C.S. & Moras, D. (2000) Zinc ion me di - ated amino acid discrimination by threonyltrna synthetase [see com ments]. Nature Struct. Biol. 7, Sankaranarayanan, R., Dock-Bregeon, A.C., Romby, P., Caillet, J., Springer, M., Rees, B., Ehresmann, C., Ehresmann, B. & Moras, D. (1999) The struc ture of threonyl-trna synthetase-trna(thr) complex enlightens its repressor ac tiv ity and re veals an es sen tial zinc ion in the ac tive site. Cell 97, Sankaranarayanan, R. & Moras, D. (1999) Mod u - lar or ga ni za tion of aminoacyl-trna syn the tas - es; in Perspectives in Struc tural Bi ol ogy (A vol - ume in hon our of Prof. G.N. Ramachandran (M. Vijayan, N.Y. & Kolaskar, A.S., eds.) pp , Universities Press (India) Limited, Hyderabad. Schimmel, P., Giege, R., Moras, D. & Yokoyama, S. (1993) An op er a tional RNA code for amino ac - ids and possible re la tion ship to ge netic code. Proc. Natl. Acad. Sci. U.S.A. 90, Schimmel, P. & Söll, D. (1979) Aminoacyl-tRNA syn the tas es: Gen eral fea tures and rec og ni tion of transfer RNAs. Annu. Rev. Biochem. 48, Schmidt, E. & Schimmel, P. (1995) Residues in a class I trna synthetase which de ter mine se - lec tiv ity of amino acid rec og ni tion in the con - text of trna. Biochemistry 34, Silvian, L.F., Wang, J. & Steitz, T.A. (1999) In - sights into editing from an Ile-tRNA syn - thetase struc ture with trna Ile and mupirocin. Science 285,

13 Vol. 48 R. Sankaranarayanan and D. Moras 335 Starzyk, R.M., Webster, T.A. & Schimmel, P. (1987) Ev i dence for dis pens able se quences in - serted into a nucleotide fold. Science 237, Suddath, F.L., Quigley, G.J., McPherson, A., Sneden, D., Kim, J.J., Kim, S.H. & Rich, A. (1974) Three-dimensional structure of yeast phenylalanine transfer RNA at 3.0 Å resolu - tion. Nature 248, Tsui, W.C. & Fersht, A.R. (1981) Probing the prin - ci ples of amino acid se lec tion using the alanyl-trna synthetase from Esch e richia coli. Nu cleic Acids Res. 9, Yarus, M. (1972) Phenylalanyl-tRNA synthetase and isoleucyl-trna Phe : A possible ver i fi ca - tion mechanism for aminoacyl-trna. Proc. Natl. Acad. Sci. U.S.A. 69,