Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli

Studies on the function of initiation factor 1 (IF 1) in the formation of 30 S initiation complexes have been carried out. IF 1 appears to prevent the dissociation of initiation factor 2 (IF 2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF 2 nor does it increase the amount of IF 2 dependent fMet tRNA and GTP bound to the 30 S subunit. Bound fMet tRNA remains stable to sucrose gradient centrifugation even in the absence of IF 1. It is postulated that the presence of IF 2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S dependent GTPase activity of IF 2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF 1 could be depleted of their complement of GTP and IF 2. Such depleted initiation complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF 2 for formation of active 70 S complexes. IF 1, which is required to prevent dissociation of IF 2 from the 30 S initiation complex, is also required for release of IF 2 from ribosomes following 70 S initiation complex formation. The mechanism of the release of IF 2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF 2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF 1 and guanine nucleotides necessary for effecting the release of IF 2. IF 2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF 1.