Isolation of two initiation factors that can partially reverse the inhibition of protein synthesis due to hemin deficiency or the hemin-controlled translational repressor in rabbit reticulocyte lysates

Abstract

The high salt wash of rabbit reticulocyte ribosomes contains two separate factors which can partially reverse the inhibition of polypeptide chain initiation that results when reticulocyte lysate is incubated in the absence of hemin. These two factors, termed initiation factor (IF) 1 and IF-2, have been separated from each other by chromatography on diethylaminoethyl cellulose and then further purified on hydroxyapatite. IF-1 forms a GTP-dependent complex with methionyl-tRNA f that is retained on Millipore filters. When these factors are added to a system containing reconstituted, salt-extracted ribosomes, IF-1 promotes the binding of methionyl-tRNA f to the 40 S subunit, whereas IF-2 promotes the formation of 80 S initiation complexes from 40 S complexes. Addition of small amounts of one factor and a saturating level of the other to the unfractionated lysate and incubation in the absence of hemin produce an additive stimulation of protein synthesis. Each factor can also partially reverse the inhibitory effect of the hemin-controlled translational repressor. The implication of these findings for the mechanism of hemin control of protein synthesis in reticulocyte lysates is discussed.