Partially purified Met-tRNA(f) binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNA(f) and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg 2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg 2+ and dissociated by Co-eIF-2B at 5 mM Mg 2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNA(f) binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNA(f) x 40S x AUG complex to the 60S ribosomal subunit to form Met-tRNA(f) x 80S x AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.
|Original language||English (US)|
|Number of pages||4|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 1979|
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