Translation initiation factor eIF-2. Cloning and expression of the human cDNA encoding the gamma-subunit

• Published in: The Journal of biological chemistry Vol. 269; no. 5; pp. 3415 - 3422
• Main Authors: GASPAR, N. J, KINZY, T. G, SCHERER, B. J, HÜMBELIN, M, HERSHEY, J. W. B, MERRICK, W. C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 04.02.1994
• Subjects: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Biological and medical sciences; Blotting, Northern; Blotting, Western; cDNA; Cell Line; Cisplatin - metabolism; Cloning, Molecular; Cross-Linking Reagents; DNA Primers; DNA, Complementary - metabolism; Epoxy Compounds - metabolism; Eukaryotic Initiation Factor-2 - biosynthesis; Eukaryotic Initiation Factor-2 - chemistry; Eukaryotic Initiation Factor-2 - isolation & purification; Fundamental and applied biological sciences. Psychology; gamma chain; Gene Expression; genes; HeLa Cells; Humans; initiation factor eIF-2; Macromolecular Substances; man; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; nucleotide sequence; Peptide Elongation Factor Tu - chemistry; predictions; Protein Structure, Secondary; RNA, Messenger - isolation & purification; RNA, Messenger - metabolism; RNA, Transfer, Met - metabolism; Sequence Homology, Amino Acid; Transfection; Translation. Translation factors. Protein processing; Human; GTP; Molecular interaction; Primary structure; Genetical translation; Initiation factor eIF2; Molecular cloning; Subunit
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)41878-3

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.