FUNCTION OF INITIATION FACTOR-5 IN EUKARYOTIC INITIATION OF PROTEIN SYNTHESIS
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The eukaryotic initiation of protein synthesis proceeds in several discrete steps requiring the participation of at least 10 specific protein factors as well as GTP. The first step is the binding of initiation factor, eIF-2 to initiator Met-tRNA(,f) and GTP to form a ternary complex, eIF-2,GTP.Met-tRNA(,f). This complex is then transferred to a 40S ribosomal subunit to which an mRNA then binds to form a 40S initiation complex 40S.mRNA.eIF-2.GTP.Met-tRNA(,f). Finally, a 60S ribosomal subunit joins the 40S initiation complex to form an 80S initiation complex (80S.mRNA.Met-tRNA(,f)), that is active in peptidyl transfer. Formation of a 40S initiation complex with natural mRNA requires the participation of several protein factors, while the formation of an 80S initiation complex by the joining of a 60S ribosomal subunit to a 40S initiation complex is mediated by a specific protein factor, eIF-5.;In order to study the mechanism of joining of a 60S ribosomal subunit to a 40S initiation complex and the function of guanine nucleotide in the overall initiation process, we have purified eIF-5 from calf liver and rabbit reticulocytes. The factor, a monomeric protein of Mr = 62,000, acts catalytically in mediating the hydrolysis of GTP bound to a 40S initiation complex, with the concomitant joining of a 60S ribosomal subunit to form an 80S intiation complex. Using purified eIF-5 preparation, we have carried out studies on the formation and release of eIF-2.GDP formed during eIF-5 mediated assembly of an 80S initiation complex. Incubation of 40S initiation complex with eIF-5 in the presence or absence of 60S ribosomal subunits at 25(DEGREES)C, causes rapid and quantitative hydrolysis of ribosome-bound GTP to form an eIF-2.GDP binary complex and P(,i). Analysis of the reaction products by Sephadex G-200 gel filtration reveals that while P(,i) is released from the ribosomes, the eIF-2.GDP complex remains bound to the ribosomal initiation complex. Preliminary evidence indicates that release of the eIF-2.GDP binary complex from the ribosomes is mediated by another specific protein factor(s). These results indicate that ribosome bound eIF-2.GDP is an intermediate in polypeptide chain initiation reaction.