170 Biochimica et Biophysica Acta, 1216 (1993) 170-172 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90575 Short Sequence-Paper

Cloning of cDNA for the/3-subunit of rabbit translation initiation factor-2 using PCR

Nigel T. Price *, Len Hall and Christopher G. Proud Department of Biochemistry, University of Bristol, Bristol BS81TD (UK)

(Received 9 July 1993)

Key words: Protein synthesis; Initiation factor-2; Eukaryote; PCR; cDNA cloning; (Rabbit)

RNA was isolated from rabbit liver and used to direct the synthesis of total cDNA. Rabbit elF-2/3 transcripts were then specifically amplified by PCR and sequenced. RACE (rapid amplification of cDNA ends) was used to obtain 3' and 5' sequences. Comparison of the deduced amino acid sequence with that of human eIF-2/3 reveals a very high degree of sequence identity.

Eukaryotic initiation factor-2 (elF-2) plays a central role in the initiation of protein synthesis. This het- erotrimeric protein interacts with a number of other components including guanine nucleotides and the ini- tiator met- tRNA and is a major site for the regulation of translational initiation (for a recent review see Ref. 1). Protein sequences derived from cDNA have been published for the /3-subunit of eIF-2 from Saccha- romyces cerevisiae (the Sui3 protein; [2]) and human liver [3]. However, most structural studies on eIF-2 have been performed with the factor from rabbit reticulocytes. Several regions of the rabbit eIF-2/3 polypeptide have been sequenced (Refs. 4, 5, and our unpublished data), numbering 137 out of the total 333 amino acid residues (see Fig. 1). Only one difference to the human protein has been reported: the replacement of serine at position 218 with tyrosine in the rabbit sequence [4]. However, our protein sequencing studies found this residue to be a serine as in the human sequence (Ref. 5, and our unpublished data). More- over, we have found this serine residue to be the major site of phosphorylation of eIF-2/3 by cAMP-dependent protein kinase (unpublished data). Here we report the sequencing of a rabbit reticulocyte cDNA clone and confirm that residue 218 is a serine. No other discrep- ancies with published partial protein sequence data were found (see Fig. 1).

* Corresponding author. Fax: + 44 272 288274. The sequence reported data in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X73836.

The cloning of rabbit elF-2/~ cDNA was performed entirely using cDNA-directed PCR. Rabbit liver total cDNA was prepared by reverse transcription of polyadenylated RNA and PCR was performed on this cDNA using Taq polymerase and primers derived from the human nucleotide sequence (corresponding to re- gions A and B, see Fig. 1). The resultant rabbit eIF-2¢3 PCR fragment was cloned into pUC18. Both strands of clones derived from three independent cDNA synthe- ses and PCRs were sequenced using a custom primer walking strategy on a DuPont Genesis 2000 automated sequencer. 5' and 3 '-RACE (rapid amplification of cDNA ends) were used to confirm the primer-defined, N-terminal coding sequence and obtain the 5' and 3' noncoding sequences. 3' RACE was performed essen- tially as in Ref. 6 using the eIF-2/3 sequence-specific primer C (see Fig. 1). 5' RACE was performed essen- tially as in Ref. 7 using an eIF-2/3 sequence-specific oligonucleotide to direct cDNA synthesis (primer D, Fig. 1). RNA ligase was then used to attach an anchor oligonucleotide to the 5' end of the NaOH-treated cDNA and PCR was performed using a second elF-2/3 sequence-specific primer (primer E, Fig. 1), and a primer complementary to the anchor.

The complete cDNA and deduced amino acid se- quences are presented in Fig. 1. The mature rabbit /3-subunit is N-terminally blocked; however, the previ- ous identification of the serine following the first me- thionine residue as the major site of phosphorylation of eIF-2 by casein kinase-2 [8] shows that this is indeed the site of initiation, despite the subsequent methion- ine codons (Met-6, Met-12) being in better sequence

171

1 50 100 PRIMER A

GCTTCGCTGATGCAAGAGCCTGGTG•GGTGGAGGGAGAGATAT•AGCAAGAAC•GAGCTGTCGCGGATTCTTGGGGCTGA•CCATCCGACTTCC•GTCCGAG0CGAACCCACC•GAG•CGCAGC• ATG TCC GGG GAC Met Ser Gly Asp

I PRIMER A 150 200 PRIMER E 250

GAG ATG ATC TTT GAT CCT ACT ATG AGC AAG AAG AAA AAG AAG AAG AAG AAG CCT TTT ATG CTA GAT GAG GAA GGG GAT GCC CAG ACA GAA GAG ACC CAG CCC TTA GAA ACA AAA Glu Met l le Phe Asp Pro Thr Met Ser LYS LYS LYS LYS LYS LYS LYS LYS Pro Phe Met Leu ASD Glu Glu Glv Aso Ala Gin Tbr Glu Glu Thr Gin Pro Leu Glu Thr Lys

10 20 30 40 300 350

GAA GTG GAG CCA GAG CCA ACG GAG GAC AAA GAT GTG GAA GCT GAT GAA GAG GAC AGT AGG AAA AAA GAT GCT TCT GAT GAT CTA GAT GAT TTG AAC TTC TTT AAC CAA AAG AAA Glu Val Glu Pro Glu Pro Thr Glu ASD Lys Asp Val Glu Ala Asp Glu Glu Asp Set Arg Lys Lys ASP Ala Ser ASp ASD Leu Aso ASP Leu Ash Phe Phe Asn Gin LYS LYS

50 60 70 80 400 450

AAG AAG AAA AAA ACA AAA AAG ATA TTT GAT ATT GAT GAA GCT GAA GAA GGT GTA AAG GAT CTT AAG ATT GAA AAT GAT GTT CAA GAG CCA GCT GAA CCA GAG GAT GAC CTT GAT LYS LYS LYS LYS Thr LYS LYS l le Phe ASD l le ASD Glu A]a Glu Glu Glv Val Lys Asp Leu Lys l le Glu Ash Asp Val Gin Glu Pro Ala G]u Pro Glu Asp Asp Leu Asp

90 I00 110 500 550 PRIMER D

ATC ATG CTT GGC AAT AAA AAA AAG AAA AAG AAG AAT GTC AAG TTC CCA GAC GAG GAT GAA ATA CTA GAG AAA GAT GAA GCT TTA GAA GAC GAA GAC AGC AAG AAA GAT GAT GGA l le Met Leu Glv ASh LYS LYS LYS LYS LYS LYS Asn Val Lvs Phe Pro ASD Glu ASD Glu l i e Leu Glu Lvs ASP GIu A1a Le~ G1u ASP G]u Asp Ser Lys Lys Asp Asp Gly

120 130 140 150 600 650 700

ATC TCA TTC AGT AAC CAG ACA GGC CCT GCT TGG GCA GGC TCA GAA AGA GAC TAC ACG TAT GAG GAG CTA CTG AAT CGA GTG TTC AAC ATC ATG AGG GAA AAG AAT CCA GAT ATG l l e Ser Phe Ser Asn Gln Thr GIy Pro Ala Trp Ala Gly Ser Glu Arg Asp Tvr Thr TYr Glu Glu Leu Leu Asn Ara Val Phe Asn l l e M~ Arq Glu Lys Asn Pro Asp Met

160 170 180 190 750 800

GTT GCT GGC GAG AAA AGG AAA TTT GTC ATG AAA CCT CCA CAG GTC GTT CGA GTA GGA ACC AAG AAA ACT TCT TTT GTC AAC TTT ACA GAT ATC TGT AAA CTA TTA CAT CGT CAG Val Ala GIY Glu LYs Arg Lys Phe Val Met Lvs Pro Pro Gln Val Val Ara Val GIv Thr Lvs Lvs Thr Ser Phe Val Asn Phe Thr ASD l l e Cvs Lvs Leu Leu His Arq Gln

200 210 220 230 850 900

CCC AAA CAC CTC CTT GCA TTT TTA CTG GCT GAA TTG GGT ACA AGT GGT TCT ATA GAT GGC AAT AAC CAA CTT GTA ATC AAA GGA AGA TTC CAA CAG AAA CAA ATA GAA AAT GTC Pro Lvs His Leu Leu A}a Phe Leu Leu Ala Glu Leu Gly Thr Ser Gly Ser l l e Asp Gly Asn Asn Gln Leu Val l l e Lys Gly Arg Phe Gin Gln Lys Gln l l e Glu Ash Val

240 250 260 270 950 I000 1049

TTG AGA AGA TAT ATC AAG GAG TAT GTC ACT TGT CAC ACG TGC CGA TCA CCA GAC ACG ATC CTG CAG AAG GAC ACC CGA CTC TAT TTC TTA CAG TGC GAA ACT TGT CAT TCT CGA Leu Arg Arg Tyr l l e Lys Glu Tyr Val Thr CYS His Thr CYS Arg Ser Pro Asp Thr I~e Leu Gln Lys Asp Thr Arg Leu Tyr Phe Leu Gin CYS Glu Thr CYS His Ser Arg

280 290 300 1100 PRIMER C PRIMER B 1150

TGC TCT GTT GCC AGT ATC AAA ACT GGC TTC CAG GCT GTC ACA GGC AAG CGA GCA CAG CTC CGT GCC AAA GCT AAC TAA TTTGCTAATCACCACTGATTTTGCAAAGTGTGTTGTGGAGAGTTGGC Cys Ser Val Ala Ser l le Lys Thr Glv Phe Gin Ala Val Thr GIY Lys Arg Ala Gln Leu Arg Ala Lys Ala Asn End

310 320 330 333 1200 1250 1300

TGGACAGGTTTCCCATCAGAGTGGA•ATGCCATTGTATTAAAAGCAAGACAGAAAAGTCGCCAAGTTCTTTGGTGAGTGGTTGGTGATCGGAAA•CCTTGCAAGATGCCGATGCTCAGGCTGTTGACATACTCATTGCCTACTTTAACAAc

1350 1400 1438 TGTCAGAAAAACATAATGGGGTAAGGAGGTGCTTTTTTAAAATCGTTCATAGACTTCTGTAAAATGCAAGATAAATTAAAGTTATTATAACAGTGAAAAAAAAAAAAAAAAAA

Fig. 1. Nucleotide and deduced amino acid sequences of rabbit elF-2/3 cDNA. The coding region of this sequence was compiled from three independent PCRs, each from independent cDNA syntheses. Underlined peptides show regions of known protein sequence (Refs. 4, 5, and our unpublished data). Cysteine residues of a potential zinc finger (residues 281,284, 302 and 305) and the polylysine blocks (residues 14-21, 79-87

and 124-129) are shown in upper case italics.

contexts. This was recently confirmed for the human mRNA, where changing the first AUG to AGG pro- duced a slightly smaller protein [9].

The rabbit and human cDNA sequences show 92% conservation at the nucleotide level, with only seven amino acid differences (see Table I), confirming the high degree of sequence conservation predicted from the existing partial protein sequence data, and ob-

TABLE I

Differences between deduced amino acid sequences of rabbit and human eIF-2fl

Residue Human elF-2# Rabbit elF-2/3

31 Thr (ACC) Ala (GCC) 54 Leu (TTG) Val (GTG) 61 Thr (ACT) Ser (AGT)

105 Ser (AGT) Asn (AAT) 111 Thr (ACT) Ala (GCT) 151 Asn (AAC) Ser (AG__C) 295 Ile (ATA) Thr (ACC)

served for other translation initiation factors [4,10]. Only one of these amino acid differences forms part of a potential functional motif in the protein. The human sequence contains two putative nucleotide binding do- main motifs, NKKD (residues 151-154) and DEEG (residues 26-29) once thought to be elements of the GTP-binding domain of eIF-2 (see Ref. 5). However, the first of these is not conserved in the rabbit se- quence (SKKD), and neither is present in the yeast sequence [2] making it unlikely that the /3-subunit is involved in GTP-binding. Indeed, all the elements of a GTP-binding site have been found in the recently- cloned y-subunit of yeast eIF-2 (the GCDll gene product [11]). A full sequence for mammalian eIF-2y is not yet available: however, GTP-binding sequences have been found in pig liver eIF-2y [12]. Other inter- esting features of eIF-2fl, which are conserved in all three known sequences, are the polylysine blocks and the zinc finger-like motif (see Fig. 1), which in yeast is apparently involved in start codon recognition [2,13]. The recently cloned eIF-2-associated protein p67 [14]

172

shows homology to the N- terminal region of elF-2/3, and in par t icular contains the polylysine tracts which have not previously been seen in prote ins other than

elF-2/3. This work was suppor ted by grants from the Medical

Research Council (UK) and the Wel lcome Trust . The authors would like to thank He len Downer and Dr D.

Emery for help with au tomated D N A sequencing.

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