Mode of Action of the Heme-Controlled Translational Inhibitor: Relationship of Eukaryotic Initiation Factor 2-Stimulating Protein to Translation Restoring Factor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 78; no. 1; pp. 220 - 223
• Main Authors: Siekierka, John, Mitsui, Ken-Ichiro, Ochoa, Severo
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.01.1981; National Acad Sciences
• Subjects: Animals; Centrifugation; Chromatography; Double stranded RNA; eIF-2 Kinase; Electrophoresis; Eukaryotic Initiation Factor-2; Eukaryotic initiation factors; Gels; Gene Expression Regulation; Globins - biosynthesis; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - metabolism; Heme - analogs & derivatives; Hemin - metabolism; Peptide Chain Initiation, Translational; Peptide Initiation Factors - isolation & purification; Peptide Initiation Factors - metabolism; Phosphorylation; Protein Biosynthesis; Protein Kinases; Proteins - isolation & purification; Proteins - metabolism; Rabbits; Reticulocytes; Reticulocytes - metabolism; RNA, Transfer - metabolism; RNA, Transfer, Amino Acyl; Serum albumins; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.78.1.220

We have purified the translation restoring factor (RF) and the eukaryotic initiation factor 2 (eIF-2) stimulating protein (ESP) to near homogeneity from the postribosomal supernatant and the ribosomal salt wash, respectively, of rabbit reticulocyte lysate. They were isolated in the form of eIF-2 complexes, apparently in a 1:1 ratio. Their virtually identical NaDodSO4/polyacrylamide gel electrophoretic patterns show, in addition to the eIF-2 α (38,000), β (52,000), and γ (54,000) bands, peptide bands at approximately 80, 65, 57, 40, and 32 kilodaltons. The apparent Mrof either complex is about 450,000, whereas that of free translation restoring factor (RF) is approximately 250,000. At 0.5 mM Mg2+, both ESP and RF stimulate ternary complex (eIF-2· GTP· Met-tRNAi) formation catalytically with unphosphorylated eIF-2. Phosphorylation of the eIF-2 α subunit by preincubation with eIF-2 α kinase and ATP, which virtually blocks eIF-2-ESP interaction, results in only partial blocking of the interaction with RF. This may explain the translation restoring activity of RF.