Two Eukaryotic Initiation Factors (IF-I and IF-II) of Protein Synthesis that Are Required to Form an Initiation Complex with Rabbit Reticulocyte Ribosomes

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Abstract
The formation of an initiation complex with rabbit reticulocyte ribosomes and certain templates is dependent upon the action of two initiation factors (IF-I and IF-II) of protein synthesis isolated from 0.5 M KCl extracts of rabbit reticulocyte polysomes. IF-I mediates the GTP-dependent, but template-independent, binding of the cytoplasmic initiator transfer RNA (rabbit liver) to a 40S ribosomal subunit. In the presence of a 60S ribosomal subunit and of AUG, AUG(U)(25), or rabbit-globin messenger ribonucleoproteins (but not of rabbit-globin messenger RNAs), IF-II causes the transfer of the initiator transfer RNA to an initiation complex containing an 80S ribosome. Although 5'-guanylylmethylenediphosphonate can replace GTP as the cofactor used in binding the initiator transfer RNA to IF-I or to a 40S ribosomal subunit, the subsequent transfer of the initiator transfer RNA to an initiation complex requires GTP and cannot take place in the presence of 5'-guanylylmethylenediphosphonate. After the formation of this initiation complex, the initiator transfer RNA is bound in the P site of the 80S ribosome since exposure of the complex to puromycin releases the methionine residue from the initiator transfer RNA as methionyl-puromycin.IF-I and IF-II are dissociated from polysomes by 0.5 M KCl as a complex having a molecular weight of about 370,000, and they remain in this form during and after gel filtration. However, the two factors can be separated by appropriate step elutions from DEAE-cellulose. After separation of the two activities, the molecular weights of IF-I and IF-II are about 150,000 and 220,000, respectively.