Functional Relationships between a Reticulocyte Polypeptide‐Chain‐Initiation Factor (IF‐MP) and the Translational Inhibitor Involved in Regulation of Protein Synthesis by Haemin

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Abstract
The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added hemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to hemin-deficient lysates prevents loss of polysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggest that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IM-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehydefixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt-washed subunits, suggesting that approximately 100,000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP .cntdot. Met-tRNAf .cntdot. GTP but does impair the factor-catalyzed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.