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. 1987 May;84(10):3141–3145. doi: 10.1073/pnas.84.10.3141

Properties of a genetically engineered G domain of elongation factor Tu.

A Parmeggiani, G W Swart, K K Mortensen, M Jensen, B F Clark, L Dente, R Cortese
PMCID: PMC304824  PMID: 3554231

Abstract

The G domain of elongation factor Tu (EF-Tu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supporting the basic activities of the intact molecule (guanine nucleotide binding in 1:1 molar ratio and GTPase activity). However, it is no longer exposed to the allosteric mechanisms regulating EF-Tu. The G-domain complexes with GTP and GDP display similar K'd values in the microM range, in contrast to EF-Tu that binds GDP much more tightly than GTP. Its GTPase shows the characteristics of a slow turnover reaction (0.1 mmol X sec-1 X mol-1 of G domain), whose rate closely corresponds to the initial hydrolysis rate of EF-Tu X GTP in the absence of effectors and lies in the typical range of GTPase of the p21 protein. Of the EF-Tu ligands only the ribosome displays a clear effect enhancing the G-domain GTPase. Our results suggest that the middle and C-terminal domain play an essential role in regulating the activity of the N-terminal domain of the intact molecule as well as in the interactions of EF-Tu with aminoacylated tRNA, elongation factor Ts, and kirromycin. With the isolation of the G domain of EF-Tu, a model protein has been constructed for studying and comparing common characteristics of the guanine nucleotide-binding proteins.

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Selected References

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

  1. Arai K., Kawakita M., Kaziro Y. Studies on the polypeptide elongation factors from E. coli. V. Properties of various complexes containing EF-Tu and EF-Ts. J Biochem. 1974 Aug;76(2):293–306. doi: 10.1093/oxfordjournals.jbchem.a130571. [DOI] [PubMed] [Google Scholar]
  2. Blumenthal T., Landers T. A., Weber K. Bacteriophage Q replicase contains the protein biosynthesis elongation factors EF Tu and EF Ts. Proc Natl Acad Sci U S A. 1972 May;69(5):1313–1317. doi: 10.1073/pnas.69.5.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bocchini V., Parlato G., De Vendittis E., Sander G., Parmeggiani A. Energetic aspects of the EF-Tu-dependent GTPase activity. A study using the antibiotic kirromycin. Eur J Biochem. 1980 Dec;113(1):53–60. doi: 10.1111/j.1432-1033.1980.tb06138.x. [DOI] [PubMed] [Google Scholar]
  4. Bosch L., Kraal B., Van der Meide P. H., Duisterwinkel F. J., Van Noort J. M. The elongation factor EF-Tu and its two encoding genes. Prog Nucleic Acid Res Mol Biol. 1983;30:91–126. doi: 10.1016/s0079-6603(08)60684-4. [DOI] [PubMed] [Google Scholar]
  5. Bourne H. R. GTP-binding proteins. One molecular machine can transduce diverse signals. 1986 Jun 26-Jul 2Nature. 321(6073):814–816. doi: 10.1038/321814a0. [DOI] [PubMed] [Google Scholar]
  6. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fasano O., Bruns W., Crechet J. B., Sander G., Parmeggiani A. Modification of elongation-factor-Tu . guanine-nucleotide interaction by kirromycin. A comparison with the effect of aminoacyl-tRNA and elongation factor Ts. Eur J Biochem. 1978 Sep 1;89(2):557–565. doi: 10.1111/j.1432-1033.1978.tb12560.x. [DOI] [PubMed] [Google Scholar]
  8. Fasano O., Crechet J. B., Parmeggiani A. Preparation of nucleotide-free elongation factor Tu and its stabilization by the antibiotic kirromycin. Anal Biochem. 1982 Jul 15;124(1):53–58. doi: 10.1016/0003-2697(82)90218-4. [DOI] [PubMed] [Google Scholar]
  9. Fasano O., De Vendittis E., Parmeggiani A. Hydrolysis of GTP by elongation factor Tu can be induced by monovalent cations in the absence of other effectors. J Biol Chem. 1982 Mar 25;257(6):3145–3150. [PubMed] [Google Scholar]
  10. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  12. Halliday K. R. Regional homology in GTP-binding proto-oncogene products and elongation factors. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(6):435–448. [PubMed] [Google Scholar]
  13. Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
  14. Kramer W., Schughart K., Fritz H. J. Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments. Nucleic Acids Res. 1982 Oct 25;10(20):6475–6485. doi: 10.1093/nar/10.20.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leberman R., Egner U. Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21. EMBO J. 1984 Feb;3(2):339–341. doi: 10.1002/j.1460-2075.1984.tb01808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McCormick F., Clark B. F., la Cour T. F., Kjeldgaard M., Norskov-Lauritsen L., Nyborg J. A model for the tertiary structure of p21, the product of the ras oncogene. Science. 1985 Oct 4;230(4721):78–82. doi: 10.1126/science.3898366. [DOI] [PubMed] [Google Scholar]
  17. McGrath J. P., Capon D. J., Goeddel D. V., Levinson A. D. Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984 Aug 23;310(5979):644–649. doi: 10.1038/310644a0. [DOI] [PubMed] [Google Scholar]
  18. Möller W., Amons R. Phosphate-binding sequences in nucleotide-binding proteins. FEBS Lett. 1985 Jul 1;186(1):1–7. doi: 10.1016/0014-5793(85)81326-0. [DOI] [PubMed] [Google Scholar]
  19. Parmeggiani A., Sander G. Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2. Mol Cell Biochem. 1981 Mar 27;35(3):129–158. doi: 10.1007/BF02357085. [DOI] [PubMed] [Google Scholar]
  20. Parmeggiani A., Swart G. W. Mechanism of action of kirromycin-like antibiotics. Annu Rev Microbiol. 1985;39:557–577. doi: 10.1146/annurev.mi.39.100185.003013. [DOI] [PubMed] [Google Scholar]
  21. Reddy P., Miller D., Peterkofsky A. Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis. J Biol Chem. 1986 Sep 5;261(25):11448–11451. [PubMed] [Google Scholar]
  22. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  23. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Scolnick E. M., Papageorge A. G., Shih T. Y. Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5355–5359. doi: 10.1073/pnas.76.10.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shih T. Y., Papageorge A. G., Stokes P. E., Weeks M. O., Scolnick E. M. Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus. Nature. 1980 Oct 23;287(5784):686–691. doi: 10.1038/287686a0. [DOI] [PubMed] [Google Scholar]
  26. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Swart G. W., Kraal B., Bosch L., Parmeggiani A. Allosteric changes of the guanine nucleotide site of elongation factor EF-Tu: a comparative study of two kirromycin-resistant EF-Tu species. FEBS Lett. 1982 Jun 1;142(1):101–106. doi: 10.1016/0014-5793(82)80228-7. [DOI] [PubMed] [Google Scholar]
  28. Sweet R. W., Yokoyama S., Kamata T., Feramisco J. R., Rosenberg M., Gross M. The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature. 1984 Sep 20;311(5983):273–275. doi: 10.1038/311273a0. [DOI] [PubMed] [Google Scholar]
  29. Tamanoi F., Walsh M., Kataoka T., Wigler M. A product of yeast RAS2 gene is a guanine nucleotide binding protein. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6924–6928. doi: 10.1073/pnas.81.22.6924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Temeles G. L., Gibbs J. B., D'Alonzo J. S., Sigal I. S., Scolnick E. M. Yeast and mammalian ras proteins have conserved biochemical properties. Nature. 1985 Feb 21;313(6004):700–703. doi: 10.1038/313700a0. [DOI] [PubMed] [Google Scholar]
  31. Wierenga R. K., Hol W. G. Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature. 1983 Apr 28;302(5911):842–844. doi: 10.1038/302842a0. [DOI] [PubMed] [Google Scholar]
  32. Wolf H., Chinali G., Parmeggiani A. Kirromycin, an inhibitor of protein biosynthesis that acts on elongation factor Tu. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4910–4914. doi: 10.1073/pnas.71.12.4910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zengel J. M., Lindahl L. A secondary promoter for elongation factor Tu synthesis in the str ribosomal protein operon of Escherichia coli. Mol Gen Genet. 1982;185(3):487–492. doi: 10.1007/BF00334145. [DOI] [PubMed] [Google Scholar]
  34. la Cour T. F., Nyborg J., Thirup S., Clark B. F. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. doi: 10.1002/j.1460-2075.1985.tb03943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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