Control of globin synthesis by the haemin-controlled translational repressor in a fractionated cell-free system from rabbit reticulocytes.

Abstract

The control of protein synthesis by haemin in rabbit reticulocyte lysates is mediated by a translational repressor protein. We have developed a fractionated globin‐synthesizing system from rabbit reticulocyte lysates that is free of endogenous repressor and therefore well suited to study the action of purified translational repressor and its antagonism to haemin.The major components of this system are: (a) rabbit reticulocyte polysomes, (b) a mouse liver pH‐5 enzyme as the source of all components for peptide chain elongation, and (c) a fraction pelleted from the rabbit reticulocyte postpolysomal supernatant by ultracentrifugation (named 200 000 ×g fraction).Crude rabbit reticulocyte lysates, when stimulated to extensive globin synthesis by haemin, can be inhibited by preparationes of purified translational repressor in its haemin‐irreversible form. In contrast, globin synthesis directed by isolated polysomes is only slightly affected by the addition of purified repressor. Addition of the 200 000 ×g fraction to polysomes produces a strong stimulation of globin synthesis that is severely inhibited by the translational repressor. If the haemin‐reversible form of the repressor is used, this inhibition can be prevented by the addition of haemin. Thus, the fractionated system shows the same effects observed with crude rabbit reticulocyte lysates and, moreover, supplies a convenient tool for the purification of the translational repressor in its haemin‐reversible form.Addition of the 200 000 ×g fraction to a protein‐synthesizing system from Ehrlich ascites tumour cells reveals the existence of α‐globin mRNA within this fraction. As determined by sucrose gradient analysis, the 200 000 ×g fraction contains two major components sedimenting at 50 S and 80 S, respectively. In view of the results presented here, the nature of these two components and their relevance to the observed features of the 200 000 ×g fraction is discussed.