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

Abstract

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.