Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Apr;79(8):2537–2540. doi: 10.1073/pnas.79.8.2537

Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2.

J Siekierka, L Mauser, S Ochoa
PMCID: PMC346234  PMID: 6953412

Abstract

Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.

Full text

PDF
2537

Images in this article

2538Image
on p.2538

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benne R., Amesz H., Hershey J. W., Voorma H. O. The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA. J Biol Chem. 1979 May 10;254(9):3201–3205. [PubMed] [Google Scholar]
  2. Benne R., Wong C., Luedi M., Hershey J. W. Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. J Biol Chem. 1976 Dec 10;251(23):7675–7681. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Cherbas L., London I. M. On the mechanism of delayed inhibition of protein synthesis in heme-defecient rabbit reticulocyte lysates. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3506–3510. doi: 10.1073/pnas.73.10.3506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clemens M. J., Pain V. M., Wong S. T., Henshaw E. C. Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2. Nature. 1982 Mar 4;296(5852):93–95. doi: 10.1038/296093a0. [DOI] [PubMed] [Google Scholar]
  6. Das A., Ralston R. O., Grace M., Roy R., Ghosh-Dastidar P., Das H. K., Yaghmai B., Palmieri S., Gupta N. K. Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5076–5079. doi: 10.1073/pnas.76.10.5076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldstein J., Safer B. Use of heparin-Sepharose for the rapid isolation of initiation and elongation factors. Methods Enzymol. 1979;60:165–181. doi: 10.1016/s0076-6879(79)60014-9. [DOI] [PubMed] [Google Scholar]
  8. Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. doi: 10.1016/0304-4173(78)90009-5. [DOI] [PubMed] [Google Scholar]
  9. Miller D. L., Weissbach H. Interactions between the elongation factors: the displacement of GPD from the TU-GDP complex by factor Ts. Biochem Biophys Res Commun. 1970 Mar 27;38(6):1016–1022. doi: 10.1016/0006-291x(70)90341-4. [DOI] [PubMed] [Google Scholar]
  10. Nagata S., Motoyoshi K., Iwasaki K. Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes. J Biochem. 1978 Feb;83(2):423–429. doi: 10.1093/oxfordjournals.jbchem.a131929. [DOI] [PubMed] [Google Scholar]
  11. Ranu R. S., London I. M. Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1079–1083. doi: 10.1073/pnas.76.3.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Siekierka J., Mitsui K. I., Ochoa S. Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor. Proc Natl Acad Sci U S A. 1981 Jan;78(1):220–223. doi: 10.1073/pnas.78.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Walton G. M., Gill G. N. Nucleotide regulation of a eukaryotic protein synthesis initiation complex;. Biochim Biophys Acta. 1975 May 1;390(2):231–245. doi: 10.1016/0005-2787(75)90344-5. [DOI] [PubMed] [Google Scholar]
  14. Weissbach H., Ochoa S. Soluble factors required for eukaryotic protein synthesis. Annu Rev Biochem. 1976;45:191–216. doi: 10.1146/annurev.bi.45.070176.001203. [DOI] [PubMed] [Google Scholar]
  15. de Haro C., Datta A., Ochoa S. Mode of action of the hemin-controlled inhibitor of protein synthesis. Proc Natl Acad Sci U S A. 1978 Jan;75(1):243–247. doi: 10.1073/pnas.75.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. de Haro C., Ochoa S. Further studies on the mode of action of the heme-controlled translational inhibitor. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1741–1745. doi: 10.1073/pnas.76.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. de Haro C., Ochoa S. Further studies on the mode of action of the heme-controlled translational inhibitor: stimulating protein acts at level of binary complex formation. Proc Natl Acad Sci U S A. 1979 May;76(5):2163–2164. doi: 10.1073/pnas.76.5.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. de Haro C., Ochoa S. Mode of action of the hemin-controlled inhibitor of protein synthesis: studies with factors from rabbit reticulocytes. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2713–2716. doi: 10.1073/pnas.75.6.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES