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The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes



The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes



Journal of Biological Chemistry 253(9): 3078-3087



Eight highly purified protein synthesis initiation factors from rabbit reticulocytes stimulate the synthesis of methionyl-puromycin or globin in assays dependent on globin 9 S mRNA. The mechanism of action of the initiation factors was studied by using sucrose gradient centrifugation to measure the binding of radioactively labeled initiation factors, methionyl-tRNAf and globin 9 S mRNA to 40 S and 80 S ribosomes. The effects of leaving out one initiation factor at a time otherwise complete reaction mixtures were determined. Ternary complex formation by initiation factor eIF-2, GTP and methionyl-tRNAf is not stimulated by any of the other initiation factors. The binding of methionyl-tRNAf to 40 S ribosomal subunits requires initiation factor eIF-2 and GTP, but not mRNA. Initiation factors eIF-3 and eIF-4C are required for maximal binding; GTP can be replaced by guanyl-5'-yl methylene diphosphonate. Maximal binding of globin 9 S mRNA to 40 S ribosomal subunits requires initiation factors eIF-1, eIF-4A, and eIF-4B and hydrolysis of ATP, in addition to initiation factors eIF-2, eIF-3, and eIF-4C, GTP and methionyl-tRNAf. The 3 polypeptides of initiation factor eIF-2 and at least 8 of the 9 major polypeptides of initiation factor eIF-3 are present in the 40 S initiation complexes in amounts approximately stoichiometric with methionyl-tRNAf and globin 9 S mRNA. Initiation factors eIF-4D and eIF-5 have no effect on 40 S initiation complex formation. The binding of methionyl-tRNAf and globin 9 S mRNA to 80 S ribosomes requires initiation factor eIF-5 and the same components mentioned for 40 S initiation complex formation. However, guanyl-5'-yl methylene diphosphonate cannot replace GTP in this reaction. Initiation factors eIF-2 and eIF-3 are absent from the 80 S initiation complexes. The reaction of methionyl-tRNAf in 80 S initiation complexes with puromycin is stimulated 2-fold by initiation factor eIF-4D. A tentative pathway for the formation of 80 S initiation complexes is proposed.

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Accession: 006715122

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PMID: 641056


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