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

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.