Gene, 123 (1993) 181-186

0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/93/$06.00 181

GENE 06884

Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase

(Multi-enzyme complex; elongation factor; rat; human; deduced amino acid sequence; phosphorylation; protein degradation; recombinant DNA; phage A library)

Adrian Vilalta”, Danny Donovana, Linda Woodb, Gabriel Vogelib and David C.H. Yang”

*Department of Chemistry, Georgetown University, Washington, DC 20057, USA: and bThe Vpjoin Co., 301 Henrietta St., Kalamazoo, MI 49007, USA Tel. (616) 385-7484

Received by J.L. Slightom: 23 May 1992; Revised/Accepted: 7 August/7 September 1992; Received at publishers: 15 October 1992

SUMMARY

A fragment of the cDNA encoding a rat valyl-tRNA synthetase (Trs”“‘)-like protein was cloned from a rat cDNA library in ;Igt I 1 using an oligodeoxyribonucleotide (oligo) probe. Three independent plaque clones containing the human TrSVa' cDNA were then isolated from a lgtl0 human erythroleukemia cDNA library using the rat cDNA fragment as the hybridization probe. Sequence analyses of the cDNA fragments provided a 3.2-kb sequence with an open reading frame that contained the ‘HIGH’ synthetase signature sequence and the tRNA 3’-end-binding motif, KMSKS, and putative Val-binding motif, EWCISRQ. The sequence was extended to the 3’ end of the cDNA by the polymerase chain reaction using an internal primer and an oligo(dT) adapter. The deduced 105 1 -amino-acid sequence shares 65% identity with yeast TrsVa’, and contains a highly basic N-terminal region, a newly evolved protease-sensitive region in sequence close to the C terminus, and several sites for protein kinase C phosphorylation. A 3-kb cDNA fragment was sub-cloned into plasmid pSVL and expressed in COS-7 cells; up to a sevenfold increase in TrsVal activity was obtained. These results confirm the cloning and sequencing of a human Trs”“‘-encoding cDNA.

INTRODUCTION

In mammalian cells, nine of the twenty aminoacyl- tRNA synthetases (Trs) including arginyl-, aspartyl-, glut-

Correspondence to: Dr. D.C.H. Yang, Department of Chemistry,

Georgetown University, Washington, DC 20057, USA. Tel. (202) 687-

6090; Fax (202) 687-6209; e-mail: dyangO1 @ggumedlib.dml.georget-

own.edu

Abbreviations: aa, amino acid(s); B., Bacillus; bp, base pair(s); cDNA,

complementary DNA; eEF1, eukaryotic elongation factor 1; kb, kilo-

base(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide;

ORF, open reading frame; PCR, polymerase chain reaction; PEST,

putative proteolysis-marking sequence in protein substrate rich in Pro,

Glu, Ser and Thr; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium

dodecyl sulfate; SSC, 0.15 M NaCl/O.lS M Na,.citrate pH 7.0; SV,

simian virus; Trs, aminoacyl-tRNA synthetase(s); Trs, gene encoding

Trs; Trs”“‘, valyl-tRNA synthetase; TPCK, tosylphenylalaninechloro-

methylketone.

amyl-, glutaminyl-, isoleucyl-, leucyl-, lysyl-, methionyl- and prolyl-Trs occur as a multi-enzyme complex (Kel- lermann et al., 1982). The organization of the Trs complex has provided excellent opportunities for structural and functional studies of protein-protein interactions beyond protein quaternary structures (for a recent review see Mir- ande, 1992). Mammalian TrsVal is particularly interesting because of its unique occurrence as a high M, complex with eEF1 (Motorin et al., 1988; Bet et al., 1989). The association of TrsVa’ with eEF1 provides a model for studies of functional and structural interactions of pro- teins in the protein biosynthetic machinery. Furthermore, recent reports on the phosphorylation of mammalian TrsVal by protein kinase C (Venema et al., 1991) opens opportunities for studies of regulatory mechanisms of mammalian Trs by reversible phosphorylation.

In this paper, we report the cloning, sequencing and

182

expression of human TrsVa’ While this manuscript was being prepared, Hsieh and Campbell (1991) reported the isolation of a cDNA from the human major histocompat- ibility complex class-III gene region that shares a high degree of aa identity to sequences of TrsVal from lower eukaryotes and suggested its product as TrsVa’. Here we present both human and rat sequences and show that additional structural motifs similar to those in bacterial and yeast TrsVa’ are conserved in the mammalian en- zymes. Several new features including potential protein kinase C phosphorylation sites, a proteolytic signal PEST sequence, and newly evolved sequences are found. We have transfected COS cells with the cDNA and demon- strated the expression of TrsVa’ activity as determined by the aminoacylation activity. Preliminary results have been presented elsewhere for the human (Vilalta and Yang, 1991) and rat TrsVa’ cDNA (Vilalta et al., 1991).

RESULTS AND DISCUSSION

(a) Cloning and sequencing of a fragment of rat TrsVa’

cDNA

A lgtl 1 rat heart cDNA library (Clontech) was first screened (Vogeli and Kaytes, 1987) with a 5’-[a-32P]-la- beled oligo GV- 174 (5’-ACCCTCAAAGCTAGTAT- GAGGGAGTTAGGGCTGCTCATCTTT). The oligo was originally designed for hybridization to sequences related to the H4 region of mouse brain voltage-gated potassium channel (Temple et al., 1988). Hybridization was done at 55°C for 12 h followed by slow cooling to 37°C and continuing hybridization at 37°C for 12 h. The filters were washed in 0.1 x SSC/l % SDS at room tem- perature and were exposed to Kodak x-ray film with an intensifying screen at - 70°C. From approximately 15 positive clones, four cDNA inserts were analyzed by di-

deoxy sequencing (Sanger et al., 1977). The sequence of the insert from clone pRS-Val-R55 shared a high degree of identity with that of TrsVa’ from E. coli (Heck and Hatfield, 1988), yeast (Jordana et al., 1987) and Bacillus stearothermophilus (Borgford et al., 1987). The degree of identity is comparable to levels found between yeast or bacterial and mammalian TrsAsP (Jacobo-Molina et al., 1989) or TrsHis (T sui and Siminovitch, 1987). The se- quence of the 1.8-kb insert from this rat cDNA clone is shown in Fig. 1. Comparative analysis of this sequence is described in section d.

(b) Cloning and sequencing of human Trs”” cDNA The presumptive rat TrsVal cDNA fragment was used

to screen a lgtl0 human erythroleukemia cDNA library (gift from Paul Bray, Johns Hopkins). Thirty four positive plaques were identified after screening 600000 plaques. Ten independent positive plaques were further purified. Seven of the ten positive plaques showed an insert of 3 kb (C6.72) and one had an insert of I kb (Cl .I 4). The se- quence of the 3-kb insert was determined (Sanger et al., 1977) using synthetic oligo primers and by subcloning HindIII, NotI, PstI and SstI fragments into the sequen- cing vector pBluescriptSK. The sequencing strategy of the resulting 3271 nt is shown in Fig. 2. The sequence of the 1 kb insert (C1.14) was found to be completely con- tained within the sequence of the 3-kb insert. Unfortu- nately, further screenings of three additional cDNA libraries did not provide any additional sequence infor- mation at the 5’ end.

In order to determine the sequence of the 3’ end of the cDNA, a 1”gtll human HeLa cDNA library was screened with the nick-translated 3’ SstI fragment (Fig. 2) of the C6.72 clone. The screening yielded a new cDNA clone (H 1.11). The sequence of the insert in H 1.11 yielded addi- tional 278-nt sequence at the 3’ end with the stop codon

120 40

240 SO

360 120 480 160 600 200 720 2‘0 840 280 960 320 080 _.^

CcTCCGTATACTGCCTGAGGcccATcAGcGGAcGTGGcATTcTTGG*TGGAcAAcATccGAGAcTGGTGcATcTcTcGGcAGcTGTGGTGGGGccAccGAATcccTGccTAcTTcATcAc LRILPEAHORTUHSVHoNIROUClSRPLWUGHRlPIYFLT

TGTCCATGACCCACCGGTAcccccTGGGGAGGAcccTGATGGGcGGTAcTGGGTAAGcGGAcGcAcTGAAGcAGAGGcccGGGAGAAGGcAGc*cGGGAATTTGGAGTGTccccTGAcAA YHDPAYPPGEDPOGRYU”SG~T~A~A~~~AA~~~G”~~O~

GATCRGCCTTCAGCAAGATGAGGATGTGTT*GAcAccTGGTTcTcGTcTGGTcTcTTTcccTTcTccATcTTTGGcTGGcccAATc*GTcAGAAGATcTcAGTGTGTTcTAcccTGGGAc I*LOOoEo”LoTVFSSGL~PF51~GUPNoSEoLS”F”PGT

CCTGCTGGACACGGGCCATGATATccT‘TTcTTcTGGGTGGcccG*ATGGTcATGcTTGGccTGAAGcTcAcTGGGAAGcTGcccTTcAGAGAGGTcTAccTccATGcAATcGTGcGGGA 1 L L E T G” 0 I L f f u Y A R H”” L G I. K L T G I: L P F R E” Y L H * I “R 0 50”

CGCTCATGGCCGGAAGATGAGcAAGTcTcTcGGcAATGTcATcGAcccccTGGATGTcA,ccATGGAGTTTccTTGcAGGGccTccATGAccAAcTAcTGAAcAGc*AccTGGATccc*G ,200 AHGRKHSYSLGN”loPLo”lHGYSLPGLHoaLLNSNLoPS 400

TGAGGTGtAGAA*CCCA*AGAAGGAcAGAGGGccGAcTTTccAGc*GGGATTccAG*GTGTGGcAcTGATGcccTAcGcTTTGGAcTcTGTGccT*cAcATcccA*GGTcGAGAcATT*A 1320 E”EKAKEGPRAoFPAG,PECGToA~~~G~cA”T~oG~o,~ 440

CCTGGATGTGAACAGG*TCCTGGGGTAccGTcAcTTcTGcAAcAA*cTcTGGAATGccAccAAGTTTGcTTTGcGcGGccTTGGGA*GGGcTTTGTAcccTcAccA*ccTccA*GccGGA 14LO iO”HRILGIRHFCNILVNAT~~A~~G~G~G~“~~~T~~~~ 480

AGGGCACGAGAGCCTGGTGGACCGCTGGATCCGCAGCCGGTTGGCAGAGGCCGTGAGGCTCAGCAATGAAGGCTTCCAGGCTTATGATTTCCCAGCCGTCACCACTGCCCAGTACAGCTT 1560 GHESLVDRVlRSRLAEAYRLSNEtFPIlDFPAYTTIOISF 520

TTGGCTCTATGAGC,CTGTGATGTCTACTTGGAGTGCCTAAAACCCGTTCTGAATGGAG,GGACCAGGTAGCAGCAGACTGTGC,CGGCAAACCCTCTACACCTGCC,GGA,G,CGCCG, ,680 UL”ELCD”“LECLKP”LlG”OO”AAOCA~OT~~,C~O”A” 560

GCGGCTGCTCTCACCCTTCATGCCCTTTGTCACAGAGGAGCTATTCCAGAGGCTGCCCCGGCGGACACCAAACGCTCCTGCTAGCCTCTGTGTCCG~ATTCC ,783 RLLSPrMPFVTEELFPRLPRRTPNAPlSLCVRNS 594

Fig. 1. Partial sequence of rat Trs “” cDNA. The cDNA insert in lgtl 1 clone pRS-val-RSS was subcloned into pBluescriptSK and sequenced on both

strands using synthetic primers and the dideoxynucleotide termination method (Sanger et al., 1977). The GenBank accession No. is M98327.

183

1 kb ‘2 Lb 3 kb

H1.11 1075

G5 72 1 2984

Cl.14 904 1980

3262

R5XRat) 08.5 2655

Fig. 2. Restriction map, locations of individual clones, and nt sequenc-

ing strategy in human Trs “al cDNA clones. Numbers that flank each

cDNA clone indicate positions relative to the assembled sequence. Hori-

zontal arrows represent sequence readings.

and poly(A) signal. To further extend to the 3’ end of the cDNA, we used for PCR an internal primer (5’-C2918A- CATGTGGCTCTGGCTTCTG) and an oligo(dT) adap- tor primer for the 3’ end with cDNA from HeLa

poly(A)+RNA as template. This product was sequenced directly and revealed the poly(A) signal followed by 12 bp and a stretch of adenylate residues. The composite nt sequence of TrsVa’ thus sequenced and the deduced aa sequence are shown in Fig. 3.

(c) Expression of human TrsVa’

The 3-kb insert in the C6.72 clone was sub-cloned into the mammalian transient expression vector pSVL (Sprague et al., 1983). This vector expresses the cDNA from the first AUG codon down stream from the SV40 promoter. As shown in Fig. 4, transfection of the green monkey COS-7 cells, which constitutively produce large T antigen, with the recombinant pSVL expression vector resulted an up to sevenfold increase of TrsVa’ activity.

(d) The aa sequence analysis

The deduced aa sequence (Fig. 3) yields a protein of 123 kDa, which is 17 kDa smaller than that previously determined by SDS-polyacrylamide gel electrophoresis (Motorin et al., 1988; Bet et al., 1989). Nonetheless, it is sufficient to catalyze enzymatic valylation of tRNA (Fig. 4) and thus contains the core catalytic domain of

Fig. 3. The nt sequence of human Trs”“’ and deduced aa sequence. The asterisk marks the stop codon. The putative basic a-k

Val binding site, KMSKS region and PEST sequence are underlined in the aa sequence. The poly(A) signal is underlined in

Get IBank accession No. is M98326.

CGAGCCG,tCTAGGAGAAGTGG,TcTATAcTcAGG*GccAGGccTcTc,cTcATcAGccAGGccccG*GGcTccTGcccTcccAAAG*cAGcTGcTcAGcTc~~AGAGGc~~A 120 R*“LGE”“L”SCARPLSHoPGP~*PA~P~,AAo~~~~A~~ 40

CGGGAGMGCTAGAGI\AATTCCAACAGAAGCAGAAGATCCAACAGCAGCAGCCACCTCCAGGGGAG~GAAACCAA~CCAGAGAAGAGGGAGAAACGGGA,CC,GGGG,CATTACCTAT 240 PEYLE~FOOKOYlOOOOPPPG~~~~~~~~~~~~O~G",TT 80

WCCTCCCIACCCACCCCGtGG***AGAAAGATGTC*GTGGCCCCATGCCCGACTCCTAC*GCCCTCGGTA,GTGGAGGC,GCCTGG,ACCCTTGG,GGGAGCAGCAGGGC,TCTTCAAG 3M) OLPT"PGE~KOYSGP~POSY~~~""~AA~"~"~~OOG~~~ (20

CCAGAGTATOGGCTCCTMTGTGTCAGCAAAATCCCCGAGGTGTCTTCATGATGTGCATCCCACCCCCC~TGTGACAOGCTCCCTGCACCTOGGCCATGCACTCACC~CGCCATC~G 480 PEYoLL~COONPRGYFMHCIPPPNVTGSLHLGHlLTYIlP 160

GACTCCCTGI\CTCG*TGGCACCGCATGCGTGGGGAGACCACCCTGTGGAACCCTGGCTGTGACCATGCAGGTATTGCCACCCAGGTGG,OG*G~GAAGAAGCTATGGCGTGACGAGG~ 600 oSLTRYHRnRGET*LUNPGCo"AG,ATO"""~~~~~~o~G 200

CTOAGCCGGCACCAGCTGGTTGAGGCCTTTCTACAGGAAGTCTGGAAGTGGAAGGAGGA~AAGGTGACCGGATTTACCACCAGTT~AG~OCTTGGCAGCTCCTTG~CTGG~TCGA 720 LSRHPL"EAFLOE"VYVIEEItDRllnPLKKLGSSLDYDP 240

GCCTGTTTCACCATGGACCCTAA*CTCTCAGCAGCTGTG*CAGAGGCCTTTGTCCGGCTTCACGAGG~GGCATC*TCTATCGCAGTACCCGCCTTGTTAACTGGTCCTGCACCCT~C 040 ACFT~OPKLSAA"TEA~YRLHEEtll"RSTRL"YYSCTLY 280 TCCGCCATCTCTGACATTGAttTGGATAAGAAGGAGCTGACAGGTCGCACCCTGCTCTCCOTGCCTGGCTAC~GGAGAAGGTGOAGTTCGGGOTCCTCOTGCCCTTTGCCTAT~GGTC 960 SA,SO,E"O~KELTGR,LLS"~G"~~~"~~G"~"~~A"~" 320

CAAGGCTCAGATAGCGACGAGGAGGTCtCAACAACTCGGATCGAGACAATGCTGGGAGATGTGGCTGTAGCTGTGCACCCCAAAGATACCAGATACCAOCACCTGMGGGGAAGlO~O OGSOSOEEY"YATTRlETHLGO"A"A""~~OT~"O"~~G~ 360

LJICCTGATCCACCCATTCCTGTCTCGGAGCCTTCCCATTGTCTTCGATGAATTTGTGGACATGGACTTTGGCACAGGTGCTGTGAAGATCACCCCCGCACATGACC~T~CTAT~i200 Y"l"PFLSRSLPI"~OE~YO~O~GTGA"~,T~A"OO~OT~ 400

OTTGCGCAGCGGCACGGGClGGAGGCAATCAGCATCATGGACTCCCGGGGGGCCCTCATCMTGTGCCTCCGCCTTTCCTGGGCCTGCCCAGGTTTGAGGCCAGG~GCGOlGCTGGTG~32O "GOR"GLEAISIHOSREALI~"~~~~~G~~A~~AA~A"~" 440

GCGCT~AGGAGCGGGGACTGTTCCGTGGCATTGAGGACAACCCCATGGTGGTGCCACTTTGCAACCGGTCGAAGGACGTGGTAGAGCCTCTGCTGCGGCCGCAGTGGTACGTTCGCTGCl440 ALKERGLFRGIEONPHYYPLC~A~~O""~~~~~~O~""~C 480

GGGWGATGGCCCAGGCTGCCAGCGCCGCTGTGACTCGGGGTGACCTCCGCATCCTGCCTGAGGCCCATCAGCGCACATGGCATGCCTGGATGGACAACATCCGGGAGTGGTGCATTTCCl56O GEHAOAASAAYTRGOLRILPEIHaRTVnlUnDHlREYClS 520

AGGCAGCTGTGGTGGGGCCATCGCATCCCAGCCTACTTTGTCACTGTCAGTGACCCAGCGGTGCCCCCTGGGGAGGACCCTGATGGGCGGTACTGGGT~GTGGACGCAATGAGGCGGAGl68O ~LUUGHR,PA"~YTYSOPAYPPGEDPDGRYYVSGRWElE 560 GCCCGGGAGAAGGCAGCCAAGGAGTTCGGAGTGTCCCCTGACAAGATCAGTCTCCAGCAAGATGAGGATGTATTGGATACCTGGTTCTCCTCTGGCCTCTTCCCCTTATCCATTTTGGGCl8OO ARE~AAKEFG"SPOKISLOOO~O"~OT~~~~G~~~~~,~G 600

TGGCCAAACCAGTCAGAAGACCTGAGTGTGTTCTACCCCGGGACACTGCTGGAGACCGGTCATGACATCCTCTTCTTCTGGGTGGCCCGGATGGTCATGCTGGGCCTG~GCTCACGGGCl92O YPWOSEOLS"F"PGTLLETG"O,~~~~"A~""~~G~~~TG 640

AGGCTGCCCTTTAGAGAGGTCTACCTCCATGCCATCGTGCGAGATGCTCACGGCCGGAAGATGAGCAAGTCTCTAGGCAATGTCATCGATCCCCTGGATGTCATCTATGGAATCTCCCTG2O4O RLP~REY"LHAI"ROAHGRKnSKSLGYVIDPLDYIIGISL 680

CACAGGAACCTCCACAACCAGC,GC,GAACAGCAACCTGGA,CCCAGCGAAGTGGAGAAGCCAAAGAAGAGAGACAGAAAGCTGACTCCCAGCGGGATTCCTGAATGTGGCACCGATGCT2160 ORNLHNOLL"SlLOPSE"EY~~~~O~~~T~GG,~~COTOA 720

CTCCGGTTTGGATTATGTGCCTACATGTCCCAGGGTCGTGACATCAACClGGATGTGAACCGGATACTGGGTTACCGCCACTTCTGCAAC~GCTCTGGAATGCCACCMGTTTGCCCTT2200 LRFGLCAIMSOGROlNLO"N~,~G"~"~C~~~~"AT~~A~ 760

CGTGGCCTTGGGAAGGGTTTTCTGCCTTCACCCACCTCCCAGCCCGGAGGCCATGAGAGCCTGGTGGACCGCTGGATCCGCAGCCGCCTGACAGAGGCTGT~GGCTCAGCAATCAAGGC2400 RGLGYGFLPSPTSOPGGHESLVDRUlRSRLTElYRLSWPt 800

TTCCAGGCCTACGACTTCCCGGCCGTCACCACTTCCCAGTACAGCTTGTGGCTCTATGAGCTCTGTGATGTCTACTTGGAGTGCCTGAAACCTGTACTGAATGGGGTGGACCAGGTGGCA252O ~OA"OFPA"TTSOYSLUL"ELCOVILECLYPVLHGYDPYI 840

tCTGAGTGTGCCCGCCAGACCCTGTACACTTGCCTGGACGTTGCCGTGAGGCTGCTCTCCACCTTCATCCCCTTCGTGACGGAG~GCTGTTCCAGAGGCTGCCCCGGAG~TGCCGCAA2~O AECAROTL"TCLOYAVRLLST~,~~"T~~~~O~~~~~~PO 880 GCTCCCCCTAGCCTCTGTGTTACCCCCTACCCGGAGCCCTCAGAGTGCTCCTGGAAGGACCCCGAGGCAGAAGCCGCCCTTGACGTGGCGCTAAGCATCACGCGAGCCGTCGCTCTGCGG2760 APPSLC"TPYPEPSECSWYO~~A~AA~O"A~G,TAA"A~A 920

CCCGACTACAACCTCACCCGGATCCGGCCTGACTGTTTCCTGGAAGTGGCGGATGAGGCGCACGGGCGGCCCTGGCATCGGCGGTGTCGGGCTACGTGCAGCCCTGGCCAGClGCAGGTG2~ AOIYLTRIRPOC~LEYAOEA"G~~~"~ACAATCG~GO~O" 9M) TCGTGGCTGTTCTGGCCTCGGTCCCGCCCCCAGGGTTCACATGTGGCTCTGGCTTCTGATCGCTGCTCCATCCACCTGCAGCTTCAGGGGCTGGTGGACCCTGCACGGGAGCTGGGCMG3OOO "Y L F VP R S R P 0 G 5"" A L A S 0 R C S I H L 0 L 0 G L "0 PAR E L G KlOOO

CTGCMGCCAAGCGAGTTGAGGCCCAGCGGCAGGCCCAGCGTCTGCGGGAACCGCGTGCTGCCTCGGGCTATCCTGTCAAGGTGCCGCTC~GTCCAGGAGGCA~T~GCCMGCTC3T2O L 0 A Y R" E A 0 R 0 A 0 R L R E P R A A 5 G 'I P" Y" P L E "0 E A 0 E A Y t1040

CAACAGACAGAAGCAGAGCTCAGCGAAGGTGGATGAGGCCATCGCCCTATTCCAGAAGATGCTGTGATCCCACCACCCAGCTTCACCCCTCACCCCCAGCGGCTCACCATGGGGATGGCA3240 0 0 T E A E L S E G G '1051

GCUATATTTTCCCACAAAAAAAAAAA 3271

ielix, HIGH sequc

the nt sequence. The

184

pC672

Fig. 4. Expression of Trs”“’ m COS-7 cells. The 3-kb insert from C6.72

was subcloned into pSVL (Pharmacia) at the XhoI site using a PCR

fragment amplified from I ng of C6.72 DNA with an Ml3 primer and

a complementary oligo with an XhoI site (underlined) at the 5’ end (5’-

CACCTCGAGATGCGAGCCGTGCTAGGAGAA). Confluent mono-

layer COS-7 cells were transfected with the recombinant plasmid,

pC672, using the &phosphate method (Chen and Okayama, 1988).

Three days after transfection, the cells were lysed with a Dounce homog-

enizer in the presence of protease inhibitors (0.2 mM PMSF/O.l mM

TPCKjl mM EDTA/l PM pepstatin). The supernatant of the lysate

after centrifugation at 12000 rpm for 30 min at 4°C was assayed for

aminoacylation activtiy immediately after preparation (Jacobo-Molina

et al., 1989). The average and range of activity of triplicate transfection

culture plates are shown and expressed in IO3 cpm [i4C]Val-tRNA/mg

protein.

human TrsVa’. Comparison of the human TrsVa’ aa se- quence with previously determined TrsVa’ sequences from bacteria and yeast shows a high degree of identity. The percentages of identity between the human Trsva’ aa se- quence and those of rat, yeast (Jordana et al., 1987), B. stearothermophilus (Borgford et al., 1987) and Ecoli (Heck and Hatfield, 1988) are 94, 65, 62 and 61%, respectively. Shared identity is extended throughout the entire se- quence except for the C terminus sequences of mamma- lian Trsva’, which contains newly evolved sequences (Fig. 5). The characteristic signature sequence of class-I Trs, HIGH, and the tRNA 3’-end binding motif, KMSKS, are conserved and are located at His”* and Lys66O, respectively. A putative Val binding motif, EW- CISRQ, is located at G1us16.

TrsVa’ appears to be highly susceptible to endogenous Ser proteases, since earlier attempts to purify the rat liver high M, TrsVa’ complex resulted in the purification of a monomeric form of the enzyme (Godar and Yang, 1988). Comparison of our isolation conditions with those of others (Bet et al., 1989; Motorin et al., 1991) suggests that the presence of diisopropylfluorophosphate during puri- fication is essential to preserve the high M, form of TrsVa’. Examination of human TrsVa’ for proteolytical signal se- quence, PEST, showed that RMPQAPPSLCVTPYPEP- SECSWK from aa 877 to 899 in the newly evolved domain has a high PEST score of 65 (Rogers et al., 1986).

Whether or not the PEST sequence is involved in signal- ing proteolytic cleavage of TrsVa’ is yet to be examined. Similar PEST sequences have been previously found in several mammalian Trs in the multi-enzyme complex of Trs (Jacobo-Molina et al., 1988).

Recently, Traugh and co-workers showed that TrsVa’ can be regulated by protein kinase C-dependent phos- phorylation (Venema et al., 1991). Examination of the deduced TrsVa’ aa sequence showed that there are eight sites that conform to protein kinase C phosphorylation consensus sequences (Pearson and Kemp, 1991). Five of the eight sites (Ser269, Thr294, Thr333, Serss4, Thr639) are clustered in the domain between the HIGH signature sequence and the tRNA 3’-end binding motif KMSKS. Two of the sites (Ser897 and Ser979) are located in the C-terminal newly evolved domain, and Sera97 is adjacent to the PEST sequence. How these sites are involved in the regulation of Trs by reversible phosphorylation is yet to be explored.

In the aa sequences of yeast Trs but not E. coli Trs, stretches of basic aa residues are usually found at the N- or C-terminal domains. The basic region in yeast TrsVal (Lys** to LYS~~) is conserved in the human enzyme (LYs~~ to Lys”). Secondary structural prediction by Chou and Fasman (1974) formalism suggests that the highly basic N-terminal region favors the cc-helical secondary struc- ture. The function of the highly basic region is not clear, but the resulting helix is amphiphilic with the basic resi- dues localized on one face of the helix (Fig. 6). This can conceivably contribute to the interaction of Trs with acidic surfaces such as membranes or RNAs. The higher affinity of yeast Trs compared to bacterial Trs toward heparin-Sepharose has been attributed to the presence of the basic region (Cirakoglu et al., 1985).

Cloning and sequencing of human TrsVa’ provides new approaches for further structural and functional studies of the enzyme and its interaction with eEF1. The avail- ability of temperature sensitive mutants of TrsVa’ in CHO cells (Ashman, 1978) should provide an opportunity for closer examination of the physiological roles of synthe- tases and regulation of synthetases in vivo.

(e) Conclusions

(I) The high similarity to E. coli, yeast and Bacillus TrsVa’, the presence of conserved structural motifs includ- ing HIGH, KMSKS and EWCISRQ, and the expression of Tr.sVa’ confirm that the cloned and sequenced cDNA encodes human TrsVa’.

(2) A highly basic putative amphiphilic helix is con- served at the N terminus of human TrsVa’.

(3) Five potential protein kinase C phosphorylation sites are clustered between the HIGH and KMSKS mo- tifs in human TrsVa’. Human TrsVa’ exhibits newly evolved

185

Cons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..L . ..P...... . . . . . . . . . . . ..PKT.... .KE.KK.E.L .KF...P... . . . . ..G... .KP.K..... Vrsh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..RAVLGE WLYSGARPL SHPPGPEAP. . . . . . . . . . . .ALPKTAAPL KKEAKKREKL EKFPPKOKIP WPPPPGEKK PKPEKREKDR 73 Vrsr : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vrsy MNKULNTLSK TFTFRLLNCH YRRSLPLCRN FSLKKSLTHN PVRFFKMSDL DNLPPVDPKT GEVIINPLKE DGSPKTPKEI EKEKKKAEKL LKFAAKPAKK NAAATTGASP KKPKKKKEVE 120 vrse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

COnS . . . . . . . . . . T.PGEKK... Vrsh DPGVITYDLP THPGEKK...

. . ..Pm.k.Y np..ve..uY .uUekqG.FK Pe........ .p.gvFci.i PPPNVTGsLH .GHALt.aiq DsliRy.RN. Gkt.l.w.pG .DHAGIATP. DVSGPMPDSY SPRYVEAAUY PUUEQQGFFK PEYGLLMCPQ NPRGVFMMCI PPPNVTGSLH LGHALTNAIP DSLTRUHRMR GETCTLYNPG CDHAGIATPV 189

vrsr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...*.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*...... . . . . . . . . . . . . . . . . . . . . Vrsy P..IPEFIDK TVPGEKKILV SLDDPALKGY NPANVESSUY DUUIKTGVFE PEFT.ADGKV KPEGVFCIPA PPPNVTGALH IGHALTIAIP DSLIRYNRMK GKTVL.FLPG FDHAGIATPS 236 vrse . . . . . . . . . . . . . . . . . . . . . . . ..MEKTY NWDIEPPLY EHYEKPGYFK PN . . . . ..GD ESPESFCIMI PPPNVTGSLH MGHAFQPTIM DTMIRYQRMQ GKNTL.UQVG TDHAGIATQM 88

Cons VVEkkiwa.e gktRH....E af....vUEU KeE.g.rI.. P..kLG.S.D U.Re.FTmdp .Ls.aV.EaF VRLh.Eg.IY R..RLVNUS. kLntAISDLE Venke.kgrt .Lsv.gykek Vrsh VVEKKLURDE GLSRH9LV.E AFLaE.VUKU KEEKGDRIYH PLKKLGSSLD WDRACFTMDP KLSAAVTEAF VRLHEEGIIY RSTRLVNUSC TLNSAISDIE VDKKELTGRT LLSV.GYKEK 307 Vrsr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LSVPGYKEK 9 VrsY VVEKQIUAKD RKTRHDYGRE AFVGK.VUEU KEEYHSRIKN PIQKLGASYD USREAFTLSP ELTKSVEEAF VRLHDEGVIY RASRLVNYSV KLNTAISNLE VENKDVKSRT LLSVPGYDEK 354 Vrse VVERKIAAEE GKTRHCDYGA EAFIDKIUEU KAESGGTITR QMRRLGNSVD UERERFTMDE GLSNAVKEVF VRLYKEDLIY RGKRLVNH)P KLRTAISDLE VENRESKGSM . . . . . . . . . . 197

Cons vefgvlvsfA ykvqgsdsde e.vvATTR.E TmLGD.aVAV hPkDpRY.hL kGK.vihPfL sRs.PlvfD. e.vDNefGTG aVKlTPAHDq NDYevG.RH. LeaI.I..sd Gal.e..... Vrsh VEFGVLVRFA YKVPGSDSDE EVVVATTRIE THLGDVAVAV HPKDTRYPHL KGKNVIHPFL SRSLPIVFD. EFVDMDFGTG AVKITPAHDP NDYEVGPRHG LEAISIMDSR GAL.I..... 420 VrSr VEFGVLVSFA CKVPGSDSDE EVVVATTRIE TMLGDVAVAV HPKDPRYQHL KGKSWHPFL SRSLPIVFD. DFVDMEFGTG AVKITPAHDQ NDYEVGPRHR LEAISIMDSK GAL.V..... 122 VrSy VEFGVLTSFA YPVIG..SDE KLIIATTRPE TIFGDTAVAV HPDDDRYKHL HGKFIaHPFL PRKIPIITDK EAVDMEFGTG AVKITPAHDQ NDYNTGKRHN LEFINILTDD GLLNE..... 458 Vrse ..UHIRYPLA DGAKTADGKD YLVVATTRPE TLLGDTGVAV NPEDPRYKDL IGKYVILPLV NRRIPIVGDE HA.DMEKGTG CVKITPAHDF NDYEVGKRHA LPMINILTFD GDIRESAPVF 314

cons Vrsh

. . . . . . . . . . . . ..vpp.f. glprF.ARKa VLealke.gL frgi.dnpm. vPLcnRs.dv .EPlLrpQUy Vr.gemA.aA saaVtrGd.r iLP.ah.rt. .sbmdnI.dU CISRPLUUGH

. . . . . . . . . . . ..NVPPPFL GLP.FEARKA VLVALKERGL FRGIEDNPMV VPLCNRSKDV VEPLLRPPUY VRCGEMAPAA SAAVTRGDLR ILPEAHPRTU HAliMDNIREU CISRPLWGH 527 Vrsr . . . . . . . . . . ..CNVPPPFL GLPRFKARKA VLEALKEPGL FRGVKDNPMV VPLCNRSKDV VEPLLRPQUY VLSGENAQAA SAAVTRGDLR ILPEAHPRTU HSUMDNIRDU CISRPLH)GH 229 vrsy . . . . . . . . . . . ..ECGPEUU GMKRFDARKK VIEQLKEKNL YVGPEDNEMT IPTCSRSGDI IEPLLKPPUU VSPSEMAKDA IKVVKDGPIT ITPKSSEAEY FHULGNIPDU CISRPLUUGH 575 VrSe DTKGNESDVY SS.EIPAEFP KLERFAARKA WAAVDALGL LEEIKPHDLT VPYGDRGGVV IEPMLTDQUY VRADVLAKPA VEAVENGDIP FVPKPYENMY FSWRDIQDU CISRPLUUGH 433

Cons RiPayfitv. dpa... gedp dGrywVsGRn eaEar.kaa. efgvs..kis LqQDEDVLDT UFSSgL.pfS .LGUP.q.ed LsvFyPg.11 etGhDIlFFU vaRM.mlglk l......tg. Vrsh RIPAYFVTVS DPAVPPGEDP DGRYUVSGRN EAEAREKAAK EFGVSPDKIS LPPDEDVLDT UFSSGLFPLS ILGUPNPSED LSVFYPGTLL ETGHDILFFU VARMVMLGLK L......TGR 641 Vrsr RIPAYFITVH DPAVPPGEDP DGRYUVSGRT EAEAREKAAR EFGVSPDKIS LQPDEDVLDT UFSSGLFPFS IFGUPNPSED LSVFYPGTLL ETGHDILFFU VARNVMLGLK L......TGK 343 Vrsy RCPVYFINIE GEE... HDRI DGDYUVAGRS MEEAEKKAAA KYPNS..KFT LEQDEDVLDT UFSSGLUPFS TLGUPEKTKD METFYPFSML ETGUDILFFU VTRMILLGLK L......TGS 684 Vrse RIPAUYDE.. . . . . . . . . . . AGNVYV.GRN EDEVRKENNL . ..GA..DVV LRPDEDVLDT UFSSALUTFS TLGUPENTDA LRPFHPTSVN VSGFDIIFFU IARMIMMTMH FIKDENGKPP 535

Cons .PFreVylha .vRDahGrKM SKSLGNVIDP LDvi.GisLq ..Lh..llns nldPseveKp kk.drk.dfP .GIpecGT.D AlRFgLcAyt SqGRDINtDv nRi.GYRhFC NKlwnAtkFa Vrsh LPFREVYLHA IVRDAHGRKM SKSLGNVIDP LDVIYGISLP RNLHNPLLNS NLDPSEVEKP KKRDRKLDFP AG1PECGT.D ALRFGLCAYH SJGRDINLDV NRILGYRHFC NKLUNATKFA 759 Vrsr LPFREVYLHA IVRDAHGRKM SKSLGNVIDP LDVIHGVSLP .GLHDPLLNS NLDPSEVEKP KK.DRGPDFP AG1PECGT.D ALRFGLCAYT SPGRDINLDV NRILGYRHFC NKLUNATKFA 460 Vrsy VPFKEVFCHS LVRDAPGRKM SKSLGNVIDP LDVITGIKLD .DLHAKLLQG NLDPREVEKA KI.GPKESYP NG1PPCGT.D AMRFALCAYT TGGRDINLDI LRVEGYRKFC NKIYPATKFA 801 Vrse VPFHTVYMTG LIRDDEGPKM SKSKGNVIDP LDMVDGISLP .ELLEKRTGN HMQPPLADKI RK.RTEKPFP NGIEPHGTCD ALRFTLAALA STGRDINM)M KRLEGYRNFC NKLUNASRFV 652

Cons L.glgkgfqp sptsgp.ghe SLvdrUl.sr lte.v.lsne . ..aydFpav ttsqY.f.U. yelCDvYlEc ..kpvlnGvd qvaa.carqT LytcLdva.r L1.pf.PF.t Eet.PRlprr Vrsh LRGLGKGFLP SPTSQPGGHE SLVDRUIRSR LTEAVRLSNQ GFQAYDFPAV TTSPYSL.UL YELCDVYLEC CLKPVLNGVD PVAAECARQT LYTCLDVAVR LLSTFIPFVT EELFPRLPRR 877 Vrsr LRGLGKGFVP SPTSKPXGHE SLVDRUIRSR LAEAVRLSNE GFQAYDFPAV TTAC’YSF.UL YELCDVYLEC .LKPVLNGVD PVAADCARPT LYTCLDVAVR LLSPFMPFVT EELFPRLPRR 578 Vrsy LMRLGDDYPP PATEGLSGNE SLVEKUILHK LTETSKIVNE ALDKRDFLTS TSS1YEF.U. YLICDVYIEN SKYLIPEGSA 1EKKSAKD.T LYILLDNALK LIHPFMPFIS EEMUQRLPKR 918 Vrse LMNTEG..PD CGFNGGEMTL SLADRUILAE FNPTIKAYRE ALDSFRFDIA AGILYEFTU. NQFCDUYLEL TK.PVMNGGT EAELRGTRHT LVTVLEGLLR LAHPIIPFIT ETIUQRVKVL 768

Cons .p.a.as.cv .pyP..y..s ..D..a.aa. d..l.it..a r.lraeyNi. ..k.....fl e...e...r. . . . . . . . ..s l.......tV . . . . . . . ..P .g........ e..ihl...G Vrsh HPQAPPSLCV TPYPEPSECS UKDPEAEAAL DVALS1TR.A VALRADYNLT ..RIRPDCFL EVADEAHGRP UHRRCRATCS . . ..PGPLPV UULFUPRSRP QGSHVALASD RCSIHLaLaG 990 Vrsr TPNAPASLCV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 Vrsy STEKAASIVK ASYP.VYVSE YDDVKSANAY DLVLNITKEA RSLLSEYNIL ..K.NGKVFV ESNHEEYFKT AEDQKDSIVS LIKAIDEVTV VRD..ASEIP EGCVLPSVNP EVNVHLLVKG1032 Vrse CGITADTIML QPFP.PYDAS QVDEAALADT EULKQAIVAV RNIRAEMNIA PGK.PLELLL RGCSADRERR VNENRGFLPT LAR.LESITV LPA..DDKGP VSVTKIIDGA ELLIPH..AG 881

Cons lvd . ..Elak lqkk..ka.. . ..rie...a ..gy..kap. .v.ea...KL . . . ..t.Ael .e..A.I..L . . . . . vrsh LVDPARELGK LPAKRVEAQR PAQRLREPRA ASGYPVKVPL EVQEADEAKL Q...PTEAEL SEGG...... . . . . . 1051 vrsr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vrsy HVDIDAEIAK VPKKLEKAKK SKNGIEPTIN SKDYETKANT PAKEANKSKL D...NTVAEI EGLEATIENL KRLKL 1104 Vrse LINKEDELAR LAKEVAKIEG EISRIENKLA NEGFVARAPE AVIAKEREKL EGYAEAKAKL IEPQAVIAAL . . . . . 951

Fig. 5. Alignment of TrsVa’ aa from different sources. Multiple sequence alignment of known Trs’“’ sequences was obtained using the GCG computer

programs package: Cons (consensus), Vrsh (human), Vrsr (rat), Vrsy (yeast) and Vrse (E. coli). In the consensus sequence, capital letters indicate

conserved aa residues in all sequences, lower case letters indicate 2 or 3 sequences share the same aa residues for that position and dots for non-

conserved aa positions.

Fig. 6. Highly basic putative a-helices from human valyl-tRNA synthe-

tase. Residues Lys3s through Leus’ are projected in the a-helical wheel

form. Seven Lys residues are shown to cluster on one side of the helix.

The + and - superscripts indicate the charges in the basic and acidic

aa residues. The calculated hydrophobic moment (p,)(Jacobo-Molina

et al., 1989) for the helix is 0.27.

sequence at the C terminus, which contains potential pro- tein kinase C phosphorylation sites and a PEST sequence.

ACKNOWLEDGEMENTS

The authors thank Drs. Paul Bray, Esther Chang and H. Okayama for the human cDNA libraries, and Pauline Yang for helpful discussions. This work was supported by an NIH grant GM-25848.

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