The Interaction of Radioactive Initiation Factor IF-2 with Ribosomes during Initiation of Protein Synthesis

Abstract

SUMMARY The interaction of initiation factor IF-Z with ribosomes during initiation of protein synthesis was studied in vifro with the radioactive factor, [32P]phosphoryl initiation factor IF-Z. The binding and release of the factor were analyzed by sucrose density gradient centrifugation. The results show that: 1. Initiation factor IF-2 is part of the complete 30 S initia- tion complex formed in the presence of A-U-G, N-formyl- methionyl transfer RNA, initiation factor IF-l, and 5’-guano- sine triphosphate or 5’-guanylyl methylenediphosphonate. 2. Initiation factor IF-2 binds to 30 S ribosomes in the absence of the other components of the 30 S initiation com- plex. The binding is stabilized by the addition of 5’-guano- sine triphosphate and by low salt concentrations in the gradi- ents. 3. Initiation factors IF-l and IF-3 greatly enhance the stability of initiation factor IF-2 binding to 30 S ribosomes. It is proposed that a complex of the 30 S ribosome and the three initiation factors may be an intermediate in the forma- tion of the complete 30 S initiation complex. 4. Initiation factor IF-2 binds stoichiometrically to active 30 S ribosomes in the presence of saturating amounts of initiation factors IF-2, IF-l, and IF-3. If the binding reac- tions are treated with glutaraldehyde before gradient analysis, 2 molecules of initiation factor IF-2 are bound per 30 S ribosome. 5. Initiation factor IF-2 and iV-formylmethionyl transfer RNA bind in equal proportions to the 70 S initiation complex formed with 5’-guanylyl methylenediphosphonate. When the 70 S complex is formed with 5’-guanosine triphosphate, however, initiation factor IF-2 no longer is bound to the complex. It is suggested that the role of 5’-guanosine tri- phosphate hydrolysis is to increase the rate of ejection of initiation factor IF-2 from the 70 S initiation complex. Initiation factor IF-2 from