A Das

Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf-EIF-1-GTP complex and Met-tRNAf binding to 40S ribosomes.

Abstract The peptide chain initiation factor, EIF-2 has been partially purified from the 0.5 M KCl ribosomal wash. The molecular weight of EIF-2 is approximately 450,000. The purified EIF-2 preparation promotes the dissociation of the ternary complex, Met-tRNA f ·EIF-1·GTP in the presence of Mg ++ and is also required along with EIF-1 for AUG-directed Met-tRNA f binding to 40S ribosomes.

Protein synthesis in rabbit reticulocytes XXXI: Purification of Co-eIF-2C and studies of its roles in peptide chain initiation.

Abstract Co-eIF-2C activity has been purified from high salt wash of reticulocyte ribosomes. The purified preparation is free from detectable levels of eIF-2, Co-eIF-2A, Co-eIF-2B and RF activities. The final step in the purification process involves use of a phosphocellulose chromatography and elution of Co-eIF-2C activity from the column with a buffer containing 2 M urea. This step results in complete removal of the contaminating Co-eIF-2B activity from the purified Co-eIF-2C preparation. Upon polyacrylamide gel electrophoresis, the final Co-eIF-2C preparation shows a single protein band under non-denaturing conditions. In the presence of SDS, the gel picture shows five prominent polypeptide bands (approximate mol. wt: 100,000; 67,000; 53,000; 45,000; and 40,000) and several faint bands. Purified Co-eIF-2C preparation strongly stimulates Met-tRNA f ·40S·AUG complex formation in the presence of eIF-2 and such stimulation is almost completely inhibited by HRI plus ATP. This study thus delineates the minimum factor requirements, namely, eIF-2 and Co-eIF-2C for formation of a stable Met-tRNA f ·40S·AUG complex.

Protein synthesis in rabbit reticulocytes XXII: A heat stable dialyzable factor (EIF-1∗) modulates Met-tRNAf binding to EIF-1☆

Abstract The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA f ·EIF-1·GTP in the absence of Mg ++ and the preformed complex is stable to Mg ++ . However, with homogeneous preparations of EIF-1, addition of Mg ++ during the initial formation of the ternary complex strongly inhibits the complex formation. A heat stable dialyzable factor (EIF-1 ∗ ) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-1 ∗ restores the Met-tRNA f binding activity of EIF-1 in the presence of 1 mM Mg ++ ; in the presence of EIF-1 ∗ , Met-tRNA f binding by EIF-1 shows a sharp Mg ++ optimum around 1 mM. EIF-1 ∗ is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-1 ∗ preparation also strongly inhibits Met-tRNA f binding to EIF-1 in the absence of Mg ++ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.

Vaccinia viral core inhibits Met-tRNAf·40S initiation complex formation with physiological mRNAs

Abstract Vaccinia viral core inhibits protein synthesis in reticulocyte lysates. In partial reactions using micrococcal nuclease treated reticulocyte lysates, the viral core inhibits Met-tRNAf binding to 40S ribosomes in response to physiological mRNAs such as globin mRNA, cowpea mosaic viral RNA, and brome mosaic viral RNA but not in response to a trinucleotide codon, AUG. The core has also no effect on Met-tRNAf binding to 40S ribosomes in a partial reaction using partially purified peptide chain initiation factors and AUG codon. The present observation of preferential inhibition by vaccinia viral core of Met-tRNAf·40S initiation complex formation with physiological mRNAs and not with an artificial mRNA such as AUG codon, suggests that the viral core inhibits some step(s) in peptide chain initiation involved in the recognition of structural feature(s) unique to physiological mRNAs.