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. 1974 Dec;71(12):4986–4990. doi: 10.1073/pnas.71.12.4986

Thiostrepton Inhibition of Initiation Factor 1 Activity in Polypeptide Chain Initiation in Escherichia coli

Probir Sarkar 1, Evan A Stringer 1, Umadas Maitra 1
PMCID: PMC434024  PMID: 4612536

Abstract

Thiostrepton, a peptide antibiotic, inhibits the GTP-dependent 70S initiation complex formation (as measured by binding of fMet-tRNA to ribosomes and concomitant hydrolysis of GTP) only when initiation factor 1 is present to permit catalytic recycling of initiation factor 2 in the initiation reaction. When initiation factor 1 is absent, the binding of fMet-tRNA and GTP hydrolysis occur stoichiometrically with respect to initiation factor 2, and thiostrepton has no effect on either reaction under these conditions. Detailed analysis of this inhibition process shows that thiostrepton prevents catalytic recycling of initiation factor 2 by blocking the action of initiation factor 1, which is required for the dissociation of initiation factor 2 from the 70S initiation complex. This dissociation is necessary for the catalytic reutilization of initiation factor 2 in the initiation reaction. The antibiotic does not directly inhibit GTP hydrolysis per se in initiation.

The inhibition of fMet-tRNA binding to ribosomes by thiostrepton is also dependent on the concentration of GTP; the inhibition is most pronounced at low concentrations of GTP, but at a high molar ratio of GTP to thiostrepton, the inhibition is completely abolished.

Keywords: antibiotic, GTP hydrolysis

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Selected References

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

  1. Benne R., Naaktgeboren N., Gubbens J., Voorma H. O. Recycling of initiation factors IF-1, IF-2 and IF-3. Eur J Biochem. 1973 Jan 15;32(2):372–380. doi: 10.1111/j.1432-1033.1973.tb02619.x. [DOI] [PubMed] [Google Scholar]
  2. Brot N., Tate W. P., Caskey C. T., Weissbach H. The requirement for ribosomal proteins L7 and L12 in peptide-chain termination. Proc Natl Acad Sci U S A. 1974 Jan;71(1):89–92. doi: 10.1073/pnas.71.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CONWAY T. W., LIPMANN F. CHARACTERIZATION OF A RIBOSOME-LINKED GUANOSINE TRIPHOSPHATASE IN ESCHERICHIA COLI EXTRACTS. Proc Natl Acad Sci U S A. 1964 Dec;52:1462–1469. doi: 10.1073/pnas.52.6.1462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cabrer B., Vázquez D., Modolell J. Inhibition by elongation factor EF G of aminoacyl-tRNA binding to ribosomes. Proc Natl Acad Sci U S A. 1972 Mar;69(3):733–736. doi: 10.1073/pnas.69.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chae Y. B., Mazumder R., Ochoa S. Polypeptide chain initiation in E. coli: studies on the function of initiation factor F1. Proc Natl Acad Sci U S A. 1969 Jul;63(3):828–833. doi: 10.1073/pnas.63.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubnoff J. S., Lockwood A. H., Maitra U. Studies on the role of guanosine triphosphate in polypeptide chain initiation in Escherichia coli. J Biol Chem. 1972 May 10;247(9):2884–2894. [PubMed] [Google Scholar]
  7. Dubnoff J. S., Maitra U. Characterization of the ribosome-dependent guanosine triphosphatase activity of polypeptide chain initiation factor IF 2. J Biol Chem. 1972 May 10;247(9):2876–2883. [PubMed] [Google Scholar]
  8. Fakunding J. L., Hershey J. W. The interaction of radioactive initiation factor IF-2 with ribosomes during initiation of protein synthesis. J Biol Chem. 1973 Jun 25;248(12):4206–4212. [PubMed] [Google Scholar]
  9. Fakunding J. L., Traut R. R., Hershey J. W. Dependence of initiation factor IF-2 activity on proteins L7 and L12 from Escherichia coli 50 S ribosomes. J Biol Chem. 1973 Dec 25;248(24):8555–8559. [PubMed] [Google Scholar]
  10. Grunberg-Manago M., Dondon J., Graffe M. Inhibition by thiostrepton of the IF-2-dependent ribosomal GTPase. FEBS Lett. 1972 May 1;22(2):217–221. doi: 10.1016/0014-5793(72)80049-8. [DOI] [PubMed] [Google Scholar]
  11. Haselkorn R., Rothman-Denes L. B. Protein synthesis. Annu Rev Biochem. 1973;42:397–438. doi: 10.1146/annurev.bi.42.070173.002145. [DOI] [PubMed] [Google Scholar]
  12. Highland J. H., Lin L., Bodley J. W. Protection of ribosomes from thiostrepton inactivation by the binding of G factor and guanosine diphosphate. Biochemistry. 1971 Nov 23;10(24):4404–4409. doi: 10.1021/bi00800a009. [DOI] [PubMed] [Google Scholar]
  13. Leder P., Bursztyn H. Initiation of protein synthesis II. A convenient assay for the ribosome-dependent synthesis of N-formyl-C14-methionylpuromycin. Biochem Biophys Res Commun. 1966 Oct 20;25(2):233–238. doi: 10.1016/0006-291x(66)90586-9. [DOI] [PubMed] [Google Scholar]
  14. Lockwood A. H., Maitra U., Brot N., Weissbach H. The role of ribosomal proteins L7 and L-12 in polypeptide chain initiation in Escherichia coli. J Biol Chem. 1974 Feb 25;249(4):1213–1218. [PubMed] [Google Scholar]
  15. Lockwood A. H., Maitra U. Relation between the ribosomal sites involved in initiation and elongation of polypeptide chains. Evidence for two guanosine triphosphatase sites. J Biol Chem. 1974 Jan 25;249(2):346–352. [PubMed] [Google Scholar]
  16. Lockwood A. H., Sarkar P., Maitra U., Brot N., Weissbach H. Effect of thiostrepton on polypeptide chain initiation in Escherichia coli. J Biol Chem. 1974 Sep 25;249(18):5831–5834. [PubMed] [Google Scholar]
  17. Lockwood A. H., Sarkar P., Maitra U. Release of polypeptide chain initiation factor IF-2 during initiation complex formation. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3602–3605. doi: 10.1073/pnas.69.12.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mazumder R. Effect of thiostrepton on recycling of Escherichia coli initiation factor 2. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1939–1942. doi: 10.1073/pnas.70.7.1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller D. L. Elongation factors EF Tu and EF G interact at related sites on ribosomes. Proc Natl Acad Sci U S A. 1972 Mar;69(3):752–755. doi: 10.1073/pnas.69.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Modolell J., Cabrer B., Parmeggiani A., Vazquez D. Inhibition by siomycin and thiostrepton of both aminoacyl-tRNA and factor G binding to ribosomes. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1796–1800. doi: 10.1073/pnas.68.8.1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Modolell J., Vazquez D., Monro R. E. Ribosomes, G-factor and siomycin. Nat New Biol. 1971 Mar 24;230(12):109–112. doi: 10.1038/newbio230109a0. [DOI] [PubMed] [Google Scholar]
  22. Pestka S., Brot N. Studies on the formation of transfer ribonucleic acid-ribosome complexes. IV. Effect of antibiotics on steps of bacterial protein synthesis: some new ribosomal inhibitors of translocation. J Biol Chem. 1971 Dec 25;246(24):7715–7722. [PubMed] [Google Scholar]
  23. Pestka S. Thiostrepton: a ribosomal inhibitor of translocation. Biochem Biophys Res Commun. 1970 Aug 11;40(3):667–674. doi: 10.1016/0006-291x(70)90956-3. [DOI] [PubMed] [Google Scholar]
  24. Richman N., Bodley J. W. Ribosomes cannot interact simultaneously with elongation factors EF Tu and EF G. Proc Natl Acad Sci U S A. 1972 Mar;69(3):686–689. doi: 10.1073/pnas.69.3.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tanaka K., Watanabe S., Teraoka H., Tamaki M. Effect of siomycin on protein synthesizing activity of Escherichia coli ribosomes. Biochem Biophys Res Commun. 1970;39(6):1189–1193. doi: 10.1016/0006-291x(70)90686-8. [DOI] [PubMed] [Google Scholar]
  26. Thach S. S., Thach R. E. 1 molecule of guanosine triphosphate is present in each 30S initiation complex. Nat New Biol. 1971 Feb 17;229(7):219–221. doi: 10.1038/newbio229219a0. [DOI] [PubMed] [Google Scholar]
  27. Weisblum B., Demohn V. Inhibition by thiostrepton of the formation of a ribosome-bound guanine nucleotide complex. FEBS Lett. 1970 Dec;11(3):149–152. doi: 10.1016/0014-5793(70)80515-4. [DOI] [PubMed] [Google Scholar]
  28. Weissbach H., Redfield B., Yamasaki E., Davis R. C., Jr, Pestka S., Brot N. Studies on the ribosomal sites involved in factors Tu and G-dependent reactions. Arch Biochem Biophys. 1972 Mar;149(1):110–117. doi: 10.1016/0003-9861(72)90304-9. [DOI] [PubMed] [Google Scholar]

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