Translation initiation factor eIF-2. Cloning and expression of the human cDNA encoding the gamma-subunit.
Translation initiation factor eIF-2 is a heterotrimeric GTP-binding protein involved in the recruitment of methionyl-tRNA, to the 40 S ribosomal subunit. To complete our characterization of eIF-2, we cloned and characterized a human cDNA encoding the largest subunit, eIF-2 gamma. From limited peptide sequence data, degenerate oligo-nucleotide primers were designed to amplify a 118-base pair DNA fragment from a cDNA library. This fragment was used as a probe to screen for larger cDNAs and eventually a clone containing the complete eIF-2 gamma coding region (1416 base pairs) was identified. It encodes a 472-amino acid protein (51.8 kDa) and contains the three consensus GTP-binding elements. The protein shares strong homology to EF-Tu, GCD11 (the yeast homolog of eIF-2 gamma), and other EF-Tu-like proteins. Transfection of COS-1 cells with the cDNA results in overexpression of a 52-kDa protein which is specifically recognized by anti-eIF-2 gamma antibodies. Cross-linking experiments with diepoxybutane and trans-diaminedichloroplatinum(II) indicate that both the beta- and gamma-subunits of eIF-2 are in close proximity to methionyl-tRNAi in ternary complexes. Possession of the eIF-2 gamma cDNA will facilitate future investigations of the interactions of GTP and methionyl-tRNAi with eIF-2.
Gaspar
N J
NJ
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Kinzy
T G
TG
Scherer
B J
BJ
Hümbelin
M
M
Hershey
J W
JW
Merrick
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002945:Cisplatin / Q000378:metabolism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000906:Antibodies; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018076:DNA, Complementary; D004852:Epoxy Compounds; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; C034697:transplatin; C007366:diepoxybutane; D010444:Peptide Elongation Factor Tu; D002945:Cisplatin