Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Jan;3(1):113–120. doi: 10.1002/j.1460-2075.1984.tb01770.x

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

F J Duisterwinkel, B Kraal, J M De Graaf, A Talens, L Bosch, G W Swart, A Parmeggiani, T F La Cour, J Nyborg, B F Clark
PMCID: PMC557306  PMID: 6323160

Abstract

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

Full text

PDF
113

Images in this article

114Fig. 1.
on p.114
116Fig. 2.
on p.116
116Fig. 3.
on p.116

Selected References

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

  1. An G., Friesen J. D. The nucleotide sequence of tufB and four nearby tRNA structural genes of Escherichia coli. Gene. 1980 Dec;12(1-2):33–39. doi: 10.1016/0378-1119(80)90013-x. [DOI] [PubMed] [Google Scholar]
  2. Arai K., Clark B. F., Duffy L., Jones M. D., Kaziro Y., Laursen R. A., L'Italien J., Miller D. L., Nagarkatti S., Nakamura S. Primary structure of elongation factor Tu from Escherichia coli. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1326–1330. doi: 10.1073/pnas.77.3.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumenthal T., Carmichael G. G. RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem. 1979;48:525–548. doi: 10.1146/annurev.bi.48.070179.002521. [DOI] [PubMed] [Google Scholar]
  4. Chinali G., Wolf H., Parmeggiani A. Effect of kirromycin on elongation factor Tu. Location of the catalytic center for ribosome-elongation-factor-Tu GTPase activity on the elongation factor. Eur J Biochem. 1977 May 2;75(1):55–65. doi: 10.1111/j.1432-1033.1977.tb11503.x. [DOI] [PubMed] [Google Scholar]
  5. Douglass J., Blumenthal T. Conformational transition of protein synthesis elongation factor Tu induced by guanine nucleotides. Modulation by kirromycin and elongation factor Ts. J Biol Chem. 1979 Jun 25;254(12):5383–5387. [PubMed] [Google Scholar]
  6. Duisterwinkel F. J., De Graaf J. M., Schretlen P. J., Kraal B., Bosch L. A mutant elongation factor Tu which does not immobilize the ribosome upon binding of kirromycin. Eur J Biochem. 1981 Jun;117(1):7–12. doi: 10.1111/j.1432-1033.1981.tb06295.x. [DOI] [PubMed] [Google Scholar]
  7. Duisterwinkel F. J., de Graaf J. M., Kraal B., Bosch L. A kirromycin resistant elongation factor EF-Tu from Escherichia coli contains a threonine instead of an alanine residue in position 375. FEBS Lett. 1981 Aug 17;131(1):89–93. doi: 10.1016/0014-5793(81)80894-0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Fischer E., Wolf H., Hantke K., Parmeggiani A. Elongation factor Tu resistant to kirromycin in an Escherichia coli mutant altered in both tuf genes. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4341–4345. doi: 10.1073/pnas.74.10.4341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hofsteenge J., Vereijken J. M., Weijer W. J., Beintema J. J., Wierenga R. K., Drenth J. Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide. Eur J Biochem. 1980 Dec;113(1):141–150. [PubMed] [Google Scholar]
  12. Hol W. G., van Duijnen P. T., Berendsen H. J. The alpha-helix dipole and the properties of proteins. Nature. 1978 Jun 8;273(5662):443–446. doi: 10.1038/273443a0. [DOI] [PubMed] [Google Scholar]
  13. Holcomb G. N., James S. A., Ward D. N. A critical evaluation of the selective tritiation method of determining C-terminal amino acids and its application to luteinizing hormone. Biochemistry. 1968 Apr;7(4):1291–1296. doi: 10.1021/bi00844a007. [DOI] [PubMed] [Google Scholar]
  14. Ivell R., Fasano O., Crechet J. B., Parmeggiani A. Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli. Biochemistry. 1981 Mar 3;20(5):1355–1361. doi: 10.1021/bi00508a049. [DOI] [PubMed] [Google Scholar]
  15. Jones M. D., Petersen T. E., Nielsen K. M., Magnusson S., Sottrup-Jensen L., Gausing K., Clark B. F. The complete amino-acid sequence of elongation factor Tu from Escherichia coli. Eur J Biochem. 1980 Jul;108(2):507–526. doi: 10.1111/j.1432-1033.1980.tb04748.x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Leberman R., Antonsson B., Giovanelli R., Guariguata R., Schumann R., Wittinghofer A. A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu. Anal Biochem. 1980 May 1;104(1):29–36. doi: 10.1016/0003-2697(80)90272-9. [DOI] [PubMed] [Google Scholar]
  18. Miyajima A., Shibuya M., Kaziro Y. Construction and characterization of the two hybrid Co1E1 plasmids carrying Escherichia coli tufB gene. FEBS Lett. 1979 Jun 15;102(2):207–210. doi: 10.1016/0014-5793(79)80001-0. [DOI] [PubMed] [Google Scholar]
  19. Nakamura S., Arai K. i., Takahashi K., Kaziro Y. Alignment of the tryptic fragments and location of sulfhydryl groups of the polypeptide chain elongation factor Tu. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1418–1424. doi: 10.1016/s0006-291x(77)80137-x. [DOI] [PubMed] [Google Scholar]
  20. Ohlsson I., Nordström B., Brändén C. I. Structural and functional similarities within the coenzyme binding domains of dehydrogenases. J Mol Biol. 1974 Oct 25;89(2):339–354. doi: 10.1016/0022-2836(74)90523-3. [DOI] [PubMed] [Google Scholar]
  21. Rossmann M. G., Moras D., Olsen K. W. Chemical and biological evolution of nucleotide-binding protein. Nature. 1974 Jul 19;250(463):194–199. doi: 10.1038/250194a0. [DOI] [PubMed] [Google Scholar]
  22. Rubin J. R., Morikawa K., Nyborg J., la Cour T. F., Clark B. F., Miller D. L. Structural features of the GDP binding site of elongation factor Tu from Escherichia coli as determined by x-ray diffraction. FEBS Lett. 1981 Jun 29;129(1):177–179. doi: 10.1016/0014-5793(81)80784-3. [DOI] [PubMed] [Google Scholar]
  23. Schulz G. E., Schirmer R. H., Pai E. F. FAD-binding site of glutathione reductase. J Mol Biol. 1982 Sep 15;160(2):287–308. doi: 10.1016/0022-2836(82)90177-2. [DOI] [PubMed] [Google Scholar]
  24. Suck D., Kabsch W. X-ray determination of the GDP-binding site of Escherichia coli elongation factor Tu by substitution with ppGpp. FEBS Lett. 1981 Apr 6;126(1):120–122. doi: 10.1016/0014-5793(81)81048-4. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Van de Klundert J. A., Van der Meide P. H., Van de Putte P., Bosch L. Mutants of Escherichia coli altered in both genes coding for the elongation factor Tu. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4470–4473. doi: 10.1073/pnas.75.9.4470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van der Meide P. H., Borman T. H., Van Kimmenade A. M., Van de Putte P., Bosch L. Elongation factor Tu isolated from Escherichia coli mutants altered in TufA and tufB. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3922–3926. doi: 10.1073/pnas.77.7.3922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van der Meide P. H., Duisterwinkel F. J., De Graaf J. M., Kraal B., Bosch L., Douglass J., Blumenthal T. Molecular properties of two mutant species of the elongation factor Tu. Eur J Biochem. 1981 Jun;117(1):1–6. doi: 10.1111/j.1432-1033.1981.tb06294.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Wolf H., Chinali G., Parmeggiani A. Mechanism of the inhibition of protein synthesis by kirromycin. Role of elongation factor Tu and ribosomes. Eur J Biochem. 1977 May 2;75(1):67–75. doi: 10.1111/j.1432-1033.1977.tb11504.x. [DOI] [PubMed] [Google Scholar]
  31. Yokota T., Sugisaki H., Takanami M., Kaziro Y. The nucleotide sequence of the cloned tufA gene of Escherichia coli. Gene. 1980 Dec;12(1-2):25–31. doi: 10.1016/0378-1119(80)90012-8. [DOI] [PubMed] [Google Scholar]
  32. van Noort J. M., Duisterwinkel F. J., Jonák J., Sedlácek J., Kraal B., Bosch L. The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules. EMBO J. 1982;1(10):1199–1205. doi: 10.1002/j.1460-2075.1982.tb00013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van de Klundert J. A., den Turk E., Borman A. H., van der Meide P. H., Bosch L. Isolation and characterization of a mocimycin resistant mutant of Escherichia coli with an altered elongation factor EF-Tu. FEBS Lett. 1977 Sep 15;81(2):303–307. doi: 10.1016/0014-5793(77)80540-1. [DOI] [PubMed] [Google Scholar]
  34. van der Meide P. H., Vijgenboom E., Talens A., Bosch L. The role of EF-Tu in the expression of tufA and tufB genes. Eur J Biochem. 1983 Feb 1;130(2):397–407. doi: 10.1111/j.1432-1033.1983.tb07166.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES