The ternary complex of initiation factor IF-I, Met-tRNA Metf and GTP An aurintricarboxylate-sensitive intermediate in the initiation of eukaryotic protein synthesis

Abstract

We have previously described an initiation factor, extensively purified from 0.5 M KCl extracts of rabbit reticulocyte polysomes, which forms a ternary 1:1 complex with the initiator tRNA [Met-tRNA Met f (rabbit liver)] and GTP. We now refer to this initiation factor as IF-I. When the ternary complex was separated from GTP and [ 35S]Met-tRNA Met f by Sephadex G-100 gel filtration and used as the only source of radioactive methionine in a rabbit reticulocyte in vitro amino acid incorporation system, the transfer of [ 35S]methionine into acid-insoluble material, using A-U-G-(U) x (x = 25 ± 5) as the messenger, was greatly stimulated as compared to the transfer of [ 35S]methionine from free [ 35S]Met-tRNA Met f. Kinetic experiments now confirm that the methionine residue in the ternary complex is a preferred precursor to the N-terminal position of newly initiated polypeptide chains. The formation of the complex is inhibited by low levels of aurintricarboxylate; however, the preformed ternary complex is stable and is not dissociated by the addition of aurintricarboxylate. When the preformed ternary complex is used in the in vitro amino acid incorporation system, aurintricarboxylate inhibits the formation of acid-insoluble material, but only at levels higher than those required for the prevention of ternary complex formation. Thus one reaction in protein biosynthesis which is very sensitive to the presence of aurintricarboxylate is the formation of the ternary complex. This phenomenon may partially explain the action of aurintricarboxylate upon eukaryotic protein synthesis initiation mediated by Met-tRNA Met f.