Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates

AbstractThe recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2 · GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(α-32P). RF and eIF-2(αP) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(α-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F−, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(αP) in the absence of F−. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2 · GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF

The Effects of Hemin and Double-Stranded RNA on α and β Globin Synthesis in Reticulocyte and Krebs II Ascites Cell-Free Systems and the Relationship of These Effects to an Initiation Factor Preparation

Protein synthesis in reticulocyte lysates ceases abruptly in the absence of added hemin or in the presence of double-stranded RNA. A similar effect of double-stranded RNA is observed in Krebs II ascites cell-free systems translating exogenous globin mRNA. The shut-off of protein synthesis is due to inhibition of initiation and can be prevented or reversed by addition of the initiation factor preparation M(3). Preparations of M(1), M(2), and dissociation factor are ineffective under these conditions. The effects of added hemin, M(3), and globin mRNA on the synthesis of α and β globin chains have been studied in the reticulocyte and ascites cell extracts. When the concentration of M(3) is rate limiting, the synthesis of β chains exceeds that of α chains. When the concentration of mRNA is rate limiting, synthesis of α and β chains is more nearly equal