Isolation of a Heme-Controlled Inhibitor of Translation That Blocks the Interaction between Messenger RNA and Eukaryotic Initiation Factor 2

A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of α-over β-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 °C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNA_f^met, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself. Since there is evidence for a specific interaction between eIF-2 and mRNA during initiation of protein synthesis, inhibition of this interaction by the inhibitor described here may be involved in translational control by heme.