Regulation of protein synthesis in rabbit reticulocyte lysates: characteristics of inhibition of protein synthesis by a translational inhibitor from heme-deficient lysates and its relationship to the initiation factor which binds Met-tRNAf.

Abstract

To define the mechanism of action of the translational inhibitor (RI), RI was partially purified. The isolated RI was utilized to examine its relationship to the translational inhibitor formed in situ in heme-deficiency, some quantitative aspects of inhibition of protein synthesis and the relationship of RI concentration to the initiation factor (IF-MP) which forms a ternary complex with Met-tRNAf and GTP (IF-MP.cntdot.Met-tRNA.cntdot.GTP). The activity of isolated RI was related to the in situ heme-deficiency inhibitor by several criteria: the biphasic kinetics of inhibition manifested by RI in lysates containing optimal levels of hemin were very similar to those observed in heme-deficiency, i.e., an initial period in which several rounds of protein synthesis proceed at the control rate followed by an abrupt decline in the rate of protein synthesis; both inhibitions were accomplished by the disaggregation of polyribosomes with a concomitant increase in 80S ribosomes; and both inhibitions were reversed by IF-MP. The isolated RI blocked protein synthesis in lysates at temperatures ranging from 15.degree.-30.degree. C. Although the rate of protein synthesis was a function of the temperature of incubation, the number of rounds of protein synthesis prior to shut-off was essentially the same at various temperatures. When RI was added to lysates, at increasing intervals after the start of incubation, the period of synthesis before shut-off (lag) progressively decreased. The inhibition of protein synthesis by RI was immediately reversed by the addition of IF-MP. The extent of reversal increased with increasing concentrations of IF-MP; at low levels of RI almost complete reversal of inhibition by IF-MP was obtained. At high levels of RI which did not appreciably increase the degree of inhibition of protein synthesis, equivalent amounts of IF-MP were less effective in reversing inhibition. The inhibition of protein synthesis by the isolated inhibitor apparently involves the initiation factor IF-MP.