Translocation events in the evolution of aminoacyl-tRNA synthetases

P oc. Na l. Acad. Sci. USA Vol. 93, pp. 8485-8489, Augus 1996 E olu ion T ansloca ion e en s in he e olu ion o aminoacyl- RNA syn he ases (his idyl- RNA syn he ase/Fugu ub ipes) SYDNEY BRENNER* AND Luis M. CORROCHANO Molecula Gene ics, Depa men o Medicine, Uni e si y o Camb idge School o Clinical Medicine, Hills Road, Camb idge CB2 2QQ, Uni ed Kingdom Con ibu ed by Syndey B enne , Ma ch 19, 1996 ABSTRACT We ha e cha ac e ized hisS, he gene encod- ing he his idyl- RNA syn he ase (HisRS) om he e aodon- oid ish Fugu ub ipes. The hisS gene is abou 3.5 kbp long and con ains 13 exons and 12 in ons o 172 bp, on a e age. The Fugu hisS gene encodes a pu a i e p o ein o 519 amino acids wi h he h ee mo i s iden i ied as signa u es o class 2 aminoacyl- RNA syn he ases. A model o he shi ing o in on 8 be ween Fugu and hams e is p oposed based on he successi e appea ance o a c yp ic splicing si e ollowed by an inse ion mu a ion ha c ea ed a new accep o si e. In addi- ion, sequence compa isons sugges ha he hisS gene has unde gone a ansloca ion h ough he i s in on. As a esul , he Fugu HisRS has an N- e minal sequence ma kedly di e en om ha in he human and hams e enzymes. We p opose ha simila e en s ha e been esponsible o a ia- ions a he N- e minal end o o he aminoacyl- RNA syn- he ases. Ou analysis sugges s ha his in ol es exchanges h ough in ons o wo exons encoding an ances al 32-amino acid mo i . Aminoacyl- RNA syn he ases join an amino acid o i s co e- sponding RNA and a e esponsible o he speci ici y o he ansla ion p ocess (1, 2). Since aminoacyl- RNA syn he ases a e key componen s o he con empo a y p o ein-syn hesis appa a us, i is possible ha knowledge o he s uc u e o genes encoding hese enzymes migh con ain clues o he e olu ion o p o ein syn hesis. I is known ha many o he aminoacyl- RNA syn he ases ha e ela ed sequences, and he 20 enzymes ha e been g ouped in o wo classes based on exclusi e se s o sequence mo i s (3). Class 1 is cha ac e ized by he amino acid sequences "HIGH" and "KMSKS" and ha e a nucleo ide binding old simila o ha ound in o he nucleo ide-binding p o eins; class 2 syn he ases ha e h ee consecu i e mo i s and an ac i e si e based on a se en-s anded an ipa allel 13-shee (2, 4-7). Aminoacyl- RNA syn he ases con ain sepa able s uc u al domains wi h di e en unc ions (8). Sequence compa isons and phylogene ic analysis ha e been used o deduce he likely e olu iona y his o y o aminoacyl- RNA syn he ases and sugges s ha he con empo a y en- zymes in he wo classes migh ha e a isen by gene duplica ion om single ances o s (9). The genomic s uc u e o he genes encoding hese enzymes is likely o help in he elucida ion o hei e olu iona y o igins, and he posi ion o in ons migh e eal ela ionships no easily ound in sequence compa isons. In on posi ions a e only known o he human glu amyl-p olyl- RNA syn he ase (Glu- P oRS), a single gene encoding he wo ac i i ies in a usion p o ein (10), human yp ophanyl- RNA syn he ase (T pRS) (11), Caeno habdi is elegans his idyl- RNA syn he ase (HisRS) (12), and hams e HisRS (13). The genome o he e aodon oid ish Fugu ub ipes, wi h abou 400 Mbp and small in ons, is 7.5 imes smalle han he human genome (14). The ela i e small size o his e eb a e genome allows he easy cloning and sequencing o genes om genomic lib a ies, and genomic s uc u es can be apidly ound. To alida e his app oach o aminoacyl- RNA syn- he ases, we selec ed he HisRS encoding gene whose in on s uc u e is known in hams e and C. elegans (12, 13). We epo he genomic s uc u e o he Fugu HisRS gene. A compa ison o he in on posi ions in HisRS wi h o he aminoacyl- RNA syn he ases p o ides e idence o ela i ely ecen ansloca ion e en s in he e olu ion o hese enzymes. MATERIALS AND METHODS Cloning and Sequencing he Fugu hisS Gene. PCR (15) was pe o med wi h wo se s o nes ed p ime s designed o anneal o conse ed a eas o hisS, he gene encoding HisRS. The p ime s used we e HislF, 5'-GAYACNCCNGTNTTYGA-3', co esponding o he amino acid sequence, DTPVFE, in he human HisRS (13, 16); His1R, 5'-CCNGTRTARTARTC-3', GTYYD; His2F, 5'-TAYCAYATHGCNAARGT, YHIAKV; and His2R, 5'-TTYTTNACYKCYTCCCA-3', K/NKVE/ AEW. Taq polyme ase (Pe kin-Elme /Ce us) was used wi h 1.5 mM Mg2+ o ampli y and clone a segmen o he Fugu hisS gene, which was e i ied by sequencing. A A2001 Sau3AI Fugu genomic lib a y (14) was sc eened wi h he hisS agmen using he me hod o Chu ch and Gilbe (17). A phage designa ed AHis was ob ained ha con ained he Fugu hisS gene and was con i med by PCR. The 15 kbp o Fugu DNA om AHis was subcloned and sequenced by he chain- e mina ion me hod (18) using syn he ic oligonucleo ides as p ime s. The hisS coding sequences we e iden i ied by simila i ies wi h o he HisRS by using he p og am BLAST (19) accessed om he Human Genome Mapping P og am Resou ce Cen e (Ha - ow, U.K.). Cloning cDNAs Encoding Fugu HisRS by PCR. To al Fugu RNA isola ed om muscle was used o cDNA syn hesis as desc ibed (20). PCR was pe o med using 1-2 ,ul o he cDNA mix and se e al se o p ime s: His7W5 (5'-CATAGCGTT- TCCTGTTC-3') and His2W2 (5'-ATAAAACTCTCTATA- ACGTC-3'), His7W8 (5'-GCTGAACCTTCATCCTC-3') and His2W2, and HisAF (5'-TGACAACCCAGCCATGACCC- 3') and His6W12 (5'-TGCGAGTCTGAATGGCG-3'). The i s wo se s o p ime s we e used o ampli y he 5' end o he gene whe eas he las se was used o ampli y he 3' end. The ampli ied DNA was cloned and sequenced by he chain- Abb e ia ions: HisRS, his idyl- RNA syn he ase; GluP oRS, glu- amyl-p olyl- RNA syn he ase; T pRS, yp ophanyl- RNA syn- he ase; GlyRS, glycyl- RNA syn he ase. Da a deposi ion: The sequence epo ed in his pape has been deposi ed in he GenBank da a base (accession no. Z54243) *To whom ep in eques s should be sen a he p esen add ess: King's College, Camb idge CB2 1ST, Uni ed Kingdom. P esen add ess: Depa amen o de Gene ica, Uni e sidad de Se illa, Apa ado 1095, E-41080 Se illa, Spain. 8485 The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked "ad e isemen " in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . 8486 E olu ion: B enne and Co ochano e mina ion me hod (18) by using syn he ic oligonucleo ides as p ime s. RESULTS The simila i y be ween human and Fugu HisRS allowed he iden i ica ion o coding sequences and in ons in he hisS gene sequence. The posi ion o in ons was u he con i med by sequencing h ee o e lapping clones con aining a Fugu hisS cDNA om 407 bp ups eam o he pu a i e ini ia o me hi- onine o 65 bp downs eam o he s op codon. The cDNA clones con ained se e al in- ame s op codons ups eam o he i s me hionine, and he s op codon in he las exon, con i m- ing he coding sequence p edic ed by simila i ies wi h human and hams e HisRS (13, 16). The hisS gene comp ises 13 exons and 12 in ons in abou 3.5 kbp (Table 1). The coding sequence has a G+C con en o 48.9%, and he in onic DNA has a G+C o 36.5%. In ons in he Fugu hisS gene ollow he GT/AG ule. All in ons a e in posi ion 0 (be ween codons) excep in on 8, which is in posi ion 1, a e he i s nucleo ide o he codon. The a e age in on size is 172 bp, bu mos in ons a e abou 100 bp in leng h (Table 1). The Fugu hisS gene encodes a pu a i e p o ein o 519 amino acids wi h he h ee mo i s iden i ied as signa u es o class 2 aminoacyl- RNA syn he ases ( esul no shown). As in he hams e gene, he posi ion o in ons in he Fugu hisS gene a e loca ed close o known unc ional and s uc u al domains. The Fugu HisRS is e y simila o i s human coun e pa : 83.5% o he esidues a e simila and 72.3% a e iden ical. The simila - i ies ex end h oughou he whole p o ein, bu he amino end is ma kedly di e en be ween he wo enzymes. In e es ingly, an in on is p esen be ween he segmen o low simila i y and he es o he gene, sugges ing ha he i s exon and he es o he gene migh ha e di e en e olu iona y o igins (Fig. 1). We could no ind any signi ican simila i y be ween he amino acid sequence o he i s exon o Fugu HisRS and any o he p o ein in he P o ein Iden i ica ion Resou ce and SwissP o da a bases. DISCUSSION I we de ine he leng h o a gene om he ini ia o codon o he s op codon, hen he Fugu hisS gene is 3.5 kbp long as compa ed wi h he hams e gene which is 18 kbp in leng h (13). Bo h genes con ain 12 in ons and 13 exons. As shown in Table 1, he leng hs o he coding sequence a e simila , abou 1.5 kbp; hus he e is an eigh - old expansion o in on leng hs in he hams e gene. Mos o he in ons in he Fugu gene a e small; 10 a e 150 bp o less. O he Fugu genes con ain simila ly small P oRS-Human P oRS-D osophila T pRS-Human GlyRS-Human GlyRS-Bombyx HisRS-Human HisRS-Hams e HisRS-Fugu H03-Human Table 1. Size o exons and in ons in he HisRS encoding gene o Fugu and hams e Exon size, bp* In on size, bp No. Fugu Hams e Fugu Hams e 1 2 3 4 5 6 7 8 9 10 11 12 13 To al 105 90 120 96 126 108 99 94 128 273 117 147 57 1560 90 90 120 96 125 109 99 99 122 240 117 145 72 1524 120 240 150 7,500 105 1,800 665 1,100 102 1,000 72 120 128 350 120 2,900 80 100 129 180 86 300 306 900 2063 16,500 Hams e da a ha e been aken om Tsui and Simino i ch (13). *The limi s o he i s and he las exons co espond o he p edic ed coding sequences. The size o he las in on in hams e has been es ima ed om he o al leng h o he HisRS encoding gene (13). in ons (14, 21-23). The di e ence in he leng h o in ons can accoun o a majo pa o he o e all educ ion o genome size o Fugu. All he in ons in he Fugu hisS gene a e in he same posi ion as in he published hams e sequence (13), excep o in on 8 which is shi ed 5 bp ela i e o he hams e in on. The sequence a ound he 5'-splicing si e o in on 8 in Fugu is he same as he 3' end o hams e exon 7 (Fig. 2). We sugges ha he shi ing o in on 8 can be explained by he ollowing plausible sequence o e en s. The Fugu sequence is aken o be he ances al o m. A c yp ic mu a ion in he in on c ea ing an al e na i e dono (GT) splicing si e could be hen ollowed by an inse ion mu a ion in- he exon c ea ing a new ac i e "AG" accep o si e. I has been cus oma y, in he compa ison o in on posi ions, o assume ha in ons in di e en homol- ogous genes ha occupy posi ions close o each o he , ega d- less o phase, ep esen he same o iginal in on, one ep e- sen a i e ha ing mo ed by an unde ined p ocess o in on sliding. This has been igh ly c i icized, and hese cases ha e been used as e idence o independen inse ions (24-26). We a e o una e he e o be able o p o ide a plausible basis o an in on shi which has occu ed ela i ely ecen ly. The i s wo exons o he human HisRS gene encode a 32-amino acid epea sequence ha is also p esen a he N- e minal ends o euka yo ic HisRS, T pRS, and glycyl- -- __----------- epea -------------- * * * ** * ** * ** * * .~~~~~~~~~~. . . . . ... . . . . ....... .... 672-CTTSEDSLVLYNRVAVQGDV.VRELKAKKAPK ED VDAA VKQLLSLKAEYKEKTGQEYKPGNPPA EIGQNISSNSSASIL ESKSLYDEVAAQGEV.VRKLKAEKSPK=AK INEA VECLLSLKAQYKEKTGKEYIPGQPPL SQSSDSSPTRNSEPAGLETP EAKVLFDKVASQGEV.VRKLKTEKAPK=DQ VDIA VQELLQLKAQYKSLIGVEYKPVSATG AEDKDKKKKEKENKSEKQNK 1118-AKDELTQEINAQGEK.VRAAKGNKAAK EV IDAE VAKLLALKAKYKEVTGTDF.PVAGRG GGGGGGSAKKAPKEAQPKPA 1-MPNSEPASLLELFNSIATQGEL.VRSLKAGNASK=DE IDSA VKMLVSLKMSYKAAACEDYKADCPPG NPAPTSNHGPDATEAEEDFV 1-MDGAGAEEVLAPLRLAVRQQGDL.VRKLKEDKAPQ VD VDKA VAELKARKRVLEAKELALQPKDDIVD RAKMEDTLKRRFFYDQAFAI 1-MADPKIEEILAPLRANVKEQGDL.VRKLKEEKAPE ID IKKA VAELKTRKKILEDKELSLAPAEDLFD RAKMEDLIKRRFFYDQSFSM 1-MAERAALEELVKLQGER.VRGLKQQKASA EL IEEE VAKLLKLKAQLGPDESKQKFVLKTPK GTRDYSPRQMAVREKVFDVI 1-MASPA.LEELVLNSRHRLVRGLKQQKASA DQ=IEEE VAKLLKLKAQLGHDESKQKFVLKTPK=GTRDYSPRQMAVREKVFDVI 1-MLAMHCARVCSVLMGCRTTTRALSIRSFPGVTL AQ=IDEE VAKLLELKAHLGGDDGKHQFVLKTAK=GTRDYNPKQMAIREKVFNTI 1-MPLLGLLPRRAWASLLSQLLRPPCASCTGAVRCQ SQ=VAEA V..... LTSQLKAHQEKPNFIIKTPK GTRDLSPQHMVVREKILDLV - ----------- epea --------------- FIG. 1. A epea in se e al aminoacyl- RNA syn he ases. The symbol "*" indica es a conse ed amino acid, and he symbol " " indica es ha mos o he amino acids in ha posi ion a e conse ed among P oRS and T pRS. In ons a e ma ked by he symbol "=" and a gap in he amino acid sequence is shown wi h a do . The posi ion o he in on in he hams e HisRS gene comes om Tsui and Simino i ch (13); he in on posi ions in he human genes encoding T pRS, and GluP oRS om F olo a e al. (11) and Kaise e al. (10) espec i ely, and posi ion o he H03 in on was ob ained om GenBank (accession no. U18936). Only he las epea om he GluP oRS o D osophila is shown. P oc. Na l. Acad. Sci. USA 93 (1996) P oc. Na l. Acad. Sci. USA 93 (1996) 8487 DNA sequence a ound in on 8 o he his idyl- RNA syn he ase gene HisRS-Fugu HisRS-Hams e HisRS-Human B TATGTTGGTATGCAAGg gaa --- c g agGTGGAATGGATTTGGCTGAACGT TATGTCCAGCAGCACGG1G3g aaa-----gc ccccagGTGTGTCTGGTAGAGCAG TATGTCCAGCAACATG4GSG GTATCCCTGGTGGAACAG Model o he shi o in on 8 Taking he Fugu in on as he ances al, an A o G change c ea es a c yp ic splicing si e Y V G M Q G G M D L A E R TATGTTGGTATGCAAGg gaga --- c g agGTGGAATGGATTTGGCTGAACGT TATGTTGGTATGCAAGg gagg --- c g agGTGGAATGGATTTGGCTGAACGT An inse ion o a G c ea es a ameshi ( op) which would be le hal unless he c yp ic splicing si e becomes unc ional (bo om). Y V G M Q G G D G F G STOP TATGTTGGTATGCAAGg gagg --- c g agGTGGAGATGGATTTGGCTGAACGT TATGTTGGTATGCAAGGTGAGg --- c g agg ggagATGGATTTGGCTGAACGT Y V G M Q G E M D L A E R The esul is a 5 nucleo ide shi in he posi ion o he in on bu only a G o E mu a ion in he amino acid sequence old Y V G M Q G G M D L A E R new Y V G M Q G E M D L A E R FIG. 2. In on shi in he hisS gene. (A) Compa ison o he sequence a ound in on 8 in Fugu, human, and hams e HisRS encoding genes is shown. Lowe case le e s deno e in on sequence, and uppe case, exon sequence. The segmen o he Fugu in on 8 iden ical o hams e exon sequence is in bold ace ype. The human and hams e sequences we e ob ained om Tsui and Simino i ch (13). (B) A model o he shi o in on 8. A c yp ic splicing si e is c ea ed by an A o G mu a ion, and an inse ion o a G c ea es a ameshi ha would be le hal unless he c yp ic splicing si es becomes unc ional, esul ing in an in on shi . Nucleo ide changes a e in bold ace ype and ma ked by a ows. RNA syn he ase (GlyRS), bu absen in hei bac e ial coun- e pa s ( e s. 16, 27, and 28; Fig. 1). Se e al copies o he same epea a e also p esen a he N- e minal end o he P oRS coding egion o he GluP oRS o human and D osophila (29, 30). The p esence o a conse ed sequence in di e en ami- noacyl- RNA syn he ases sugges s ha hey had a common o igin (8, 28, 31). The DNA sequence encoding he epea is spli by an in on in he genes encoding hams e HisRS and human T pRS, and in wo o he h ee epea s o he human GluP oRS (Fig. 1). Al hough he posi ion o his in on di e s by wo codons in he P oRS and HisRS genes, we will ea i as he same in on. I is emp ing o specula e ha his in on migh also be p esen in a simila loca ion in he e eb a e genes encoding GlyRS. The unc ion o he epea is no known, bu he i s wo exons o he gene encoding human HisRS, including he epea , a e necessa y o enzyma ic ac i i y (28). Howe e , in he case o he human T pRS gene, al e na i e splicing elimina es he exon encoding he i s hal o he epea wi hou loss o unc ion (32). The p esence o he epea as well as he in on in he gene encoding T pRS, a class 1 enzyme, sugges s ha his sequence sha es a common o igin wi h he same wo exons o he genes encoding human P oRS, and HisRS class 2 enzymes. Fig. 1 also e eals ha he simila i ies be ween human P oRS and T pRS ex end beyond he epea and end a a posi ion whe e ano he in on is p esen in he human HisRS gene. We can bes explain hese esul s by assuming ha he wo-exon epea was p esen in he ances o gene ha ga e ise o a leas some, i no all, o he class 2 enzymes, and ha he T pRS gene acqui ed his sequence by ansloca ion in o he second in on o a duplica ed class 2 enzyme gene (Fig. 3). We ha e o explain he absence o his in on om he T pRS gene by i s subse- quen loss, which is mo e plausible han an accu a e usion be ween wo coding sequences. We hink hese e en s ook place a long ime ago, a leas be o e he di e gence o insec s and mammals as deduced om he simila i y o he s uc u es o P oRS and GlyRS. The amino end o he Fugu HisRS is e y di e en om he human HisRS, and i only has he second hal o he 32-amino acid epea (Fig. 1). We no e ha he i s in on clea ly sepa a es he egion o low simila i y om he es o he p o ein, sugges ing ha he i s exon in he human and Fugu hisS genes had di e en e olu iona y o igins. We p opose ha he Fugu gene has been ansloca ed h ough he i s in on om i s o iginal posi ion in o a new loca ion, cap u ing an exon and acqui ing a new sequence a i s amino end (Fig. 3). This is suppo ed by he ac ha we could no de ec any simila i y be ween he 5' ups eam egions o he human and Fugu hisS genes. Such ansloca ions may be impo an in e olu ion, allowing coding sequences o acqui e no el egula o y p op- e ies, bu hey could also be g a ui ous. In any e en , we can p edic ha he genes would occupy di e en posi ions in he human and ish genomes. An analogous ansloca ion seems o ha e occu ed in a sequence homologous o he human HisRS gene, designa ed H03 in Fig. 1, which is ound jus ups eam o he gene bu in he opposi e o ien a ion (33). This homolog has an amino end e y di e en om he o he human and hams e HisRS, wi h an in on sepa a ing egions o high and low simila i y. Like Fugu HisRS, H03 has mos likely los he amino end o he epea by ansloca ion o an in e ed duplica ion h ough he i s in on (Fig. 3). Since such a sequence was no ound in Fugu, his is also a compa a i ely ecen e en . Exon shu ling has been p oposed as a majo mode o he e olu ion o p o ein di e si y. I his happened a a e y ea ly s age o p o ein e olu ion, hen he e needs o be some co espondence be ween p imi i e exons and seconda y es- uc u e elemen s o else mos o he combina ions would be A E olu ion: B enne and Co ochano 8488 E olu ion: B enne and Co ochano hypo e ical ansloca ion e en s in he e olu ion o aminoacyl- RNA syn he ases 32-aa epea exons es o he gene in on loss ances al class 2 P olyl- RNA syn he ase gene aminoacyl- RNA syn he ase gene in e ed duplica ion and ansloca ion & I 1 / ~~~~H03 I p His idyl- RNA syn he ase gene ansloca ion h oug L 1 L IF he i s inl on E His idyl- RNA syn he ase gene Luman Fugu His idyl- RNA syn he ase gene class 2 aminoacyl- RNA syn he ase gene LIJEx. ansloca ion h ough he second in on in on loss T yp ophanyl- RNA syn he ase gene T yp ophanyl- RNA syn he ase gene FIG. 3. Hypo he ical ansloca ion e en s in he e olu ion o aminoacyl- RNA syn he ases. The wo exons encoding he 32-amino acid epea and he es o he gene a e shown by g ey and ha ched boxes, espec i ely. An ances al class 2 aminoacyl- RNA syn he ase gene con aining he wo exons encoding he 32-amino acid epea ga e ise o he genes encoding a leas P oRS and HisRS. P esen -day P oRS in animals con ain se e al copies o he epea and is used o he gene encoding GluRS (no shown). The gene encoding HisRS su e ed an in e ed duplica ion and a ansloca ion in he human lineage esul ing in wo genes in opposi e o ien a ion. The ansloca ion migh ha e occu ed h ough he i s in on esul ing in he cap u ing o a new exon in he H03 gene (HisRS homolog). In he ish lineage, a ansloca ion h ough he i s in on allowed he cap u ing o a new exon in he Fugu HisRS gene. The human T pRS gene (a class 1 enzyme) has cap u ed he wo exons epea by ansloca ion in o a class 2 gene. A u he in on loss esul ed in he p esen -day s uc u e o he human T pRS gene. useless. Whe he a esidue o his is s ill p esen in con em- po a y genes is s ill a ma e o deba e (25, 26, 34-37). Howe e , usion o la ge p o ein domains would also be acili a ed by exchanges h ough in ons, which is less demand- ing han he ecombina ion o coding sequences. The e a e many examples o his in he s uc u e o complex genes, and in many ins ances an in on can be ound o ma k he bounda y be ween wo di e en la ge domains (38). We p e e o call hese e en s ansloca ions, a he han using he loose e m o exon shu ling, since we wan o emphasize ha his is a ecombina ion p ocess which mus lead o a change in he posi ion o a gene in he genome. We also belie e ha such a ansloca ion will p o e o be he majo sou ce o e olu iona y di e si y in he e olu ion o complex o ganisms. Th ough i , no el pa hs o gene egula ion can be explo ed. Since many genes con ain in ons in hei 5' un ansla ed sequences, ansloca ion h ough hese in ons would be he bes way o achie e such ansloca ions, because he exchange p ocess need no depend on he phases o he ecombining in ons. Since he posi ion o a gene in a genome changes as a consequence o ansloca ion, he compa a i e s udy o genome s uc u es migh con ain impo an clues o he e olu ion o complex unc ions, e en hough many o hese ansloca ion e en s may no be p oduc i e bu g a ui ous. Aminoacyl- RNA syn he ases a e old p o eins and a e com- posed o se e al s uc u al and unc ional mo i s (8). We ha e ound ha gene ansloca ion appea s as he likely mechanism esponsible o he di e gence in simila i y a he amino end be ween Fugu and human HisRS. The ele ance o ansloca- ions in he e olu ion o aminoacyl- RNA syn he ases can only be assessed as mo e esul s a e ob ained on he genomic s uc u e o he genes encoding hese p o eins. 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