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 pr otein involved in the recruitment of methionyl-tRNA(i) to the 40 S rib osomal subunit. To complete our characterization of eIF-2, we cloned a nd characterized a human cDNA encoding the largest subunit, eIF-2gamma . From limited peptide sequence data, degenerate oligonucleotide prime rs were designed to amplify-a 118-base pair DNA fragment from a cDNA l ibrary. This fragment was used as a probe to screen for larger cDNAs a nd eventually a clone containing the complete eIF-2gamma 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-2gamma), and other EF-Tu-like proteins. Transfection of COS-1 cell s with the cDNA results in overexpression of a 52-kDa protein which is specifically recognized by anti-eIF-2gamma antibodies. Cross-linking experiments with diepoxybutane and trans-diaminedichloroplatinum(II) i ndicate that both the beta- and gamma-subunits of eIF-2 are in close p roximity to methionyl-tRNA(i) in ternary complexes. Possession of the eIF-2gamma cDNA will facilitate future investigations of the interacti ons of GTP and methionyl-tRNA(i) with eIF-2.