TRANSLATIONAL LEVEL CONTROL OF PROTEIN SYNTHESIS IN EUKARYOTIC CELLS

During heme-deficiency in reticulocyte lysate, a translational inhibitor (HRI, heme-regulated inhibitor) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(,f) binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors resulting in a net reduction of ternary complex (Met-tRNA(,f)(.)eIF-2(.)GTP) formation and AUG-dependent transfer of Met-tRNA(,f) to 40S ribosomal subunits. A factor (RF) that reverses protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte ribosomal salt wash and post-ribosomal supernatant. The purified RF restores protein synthesis activity of the heme-deficient lysate to the level observed in the presence of hemin. No direct correlation exists between amount of eIF-2 activity and ability to reverse protein synthesis inhibition in heme-deficient lysates. Homogeneous preparations of eIF-2 are completely inactive in reversal of protein synthesis inhibition in heme-deficient lysate. RF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. Both the ternary complex inhibition reversal and protein synthesis inhibition reversal activities co-sediment at 12.5S upon glycerol density gradient centrifugation and both activities are heat and NEM sensitive. Purified RF does not dephosphorylate phosphorylated eIF-2 (HRI-catalyzed) nor prevent the phosphorylation of eIF-2 by HRI in a fractionated system. RF stimulates ternary complex formation by both phosphorylated and non-phosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to eIF-2 kinases such as HRI may be modulated by RF