Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Feb 11;18(3):437–441. doi: 10.1093/nar/18.3.437

The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.

G Ott 1, J Jonák 1, I P Abrahams 1, M Sprinzl 1
PMCID: PMC333445  PMID: 2408011

Abstract

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.

Full text

PDF
437

Images in this article

439Image
on p.439
440Image
on p.440

Selected References

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

  1. Duffy L. K., Gerber L., Johnson A. E., Miller D. L. Identification of a histidine residue near the aminoacyl transfer ribonucleic acid binding site of elongation factor Tu. Biochemistry. 1981 Aug 4;20(16):4663–4666. doi: 10.1021/bi00519a022. [DOI] [PubMed] [Google Scholar]
  2. Duisterwinkel F. J., Kraal B., De Graaf J. M., Talens A., Bosch L., Swart G. W., Parmeggiani A., La Cour T. F., Nyborg J., Clark B. F. Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure. EMBO J. 1984 Jan;3(1):113–120. doi: 10.1002/j.1460-2075.1984.tb01770.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Holmes W. M., Hurd R. E., Reid B. R., Rimerman R. A., Hatfield G. W. Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1068–1071. doi: 10.1073/pnas.72.3.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jonák J., Karas K. Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L-phenylalanyl chloromethane abolishes its ability to interact with the 3'-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA. FEBS Lett. 1989 Jul 17;251(1-2):121–124. doi: 10.1016/0014-5793(89)81440-1. [DOI] [PubMed] [Google Scholar]
  6. Jonák J., Petersen T. E., Clark B. F., Rychlík I. N-Tosyl-L-phenylalanylchloromethane reacts with cysteine 81 in the molecule of elongation factor Tu from Escherichia coli. FEBS Lett. 1982 Dec 27;150(2):485–488. doi: 10.1016/0014-5793(82)80795-3. [DOI] [PubMed] [Google Scholar]
  7. Jonák J., Smrt J., Holý A., Rychlík I. Interaction of Escherichia coli EF-Tu.GTP and EF-Tu.GDP with analogues of the 3' terminus of aminoacyl-tRNA. Eur J Biochem. 1980 Apr;105(2):315–320. doi: 10.1111/j.1432-1033.1980.tb04503.x. [DOI] [PubMed] [Google Scholar]
  8. Jurnak F., McPherson A., Wang A. H., Rich A. Biochemical and structural studies of the tetragonal crystalline modification of the Escherichia coli elongation factor Tu. J Biol Chem. 1980 Jul 25;255(14):6751–6757. [PubMed] [Google Scholar]
  9. 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]
  10. Masuda E., Louie A., Jurnak F. Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA. J Biol Chem. 1985 Jul 25;260(15):8702–8705. [PubMed] [Google Scholar]
  11. Möller W., Schipper A., Amons R. A conserved amino acid sequence around Arg-68 of Artemia elongation factor 1 alpha is involved in the binding of guanine nucleotides and aminoacyl transfer RNAs. Biochimie. 1987 Sep;69(9):983–989. doi: 10.1016/0300-9084(87)90232-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Ott G., Faulhammer H. G., Sprinzl M. Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus. Eur J Biochem. 1989 Sep 15;184(2):345–352. doi: 10.1111/j.1432-1033.1989.tb15025.x. [DOI] [PubMed] [Google Scholar]
  14. Peter M. E., Wittmann-Liebold B., Sprinzl M. Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu. Biochemistry. 1988 Dec 27;27(26):9132–9139. doi: 10.1021/bi00426a010. [DOI] [PubMed] [Google Scholar]
  15. Pingoud A., Urbanke C. Aminoacyl transfer ribonucleic acid binding site of the bacterial elongation factor Tu. Biochemistry. 1980 May 13;19(10):2108–2112. doi: 10.1021/bi00551a017. [DOI] [PubMed] [Google Scholar]
  16. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Scolnick E. M. Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4725–4729. doi: 10.1073/pnas.83.13.4725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  18. Thompson R. C., Dix D. B., Gerson R. B., Karim A. M. A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes. J Biol Chem. 1981 Jan 10;256(1):81–86. [PubMed] [Google Scholar]
  19. Van Noort J. M., Kraal B., Sinjorgo K. M., Persoon N. L., Johanns E. S., Bosch L. Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis. Eur J Biochem. 1986 Nov 3;160(3):557–561. doi: 10.1111/j.1432-1033.1986.tb10074.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Wagner T., Sprinzl M. Enzymic binding of aminoacyl-tRNA to Escherichia coli ribosomes using modified tRNA species and tRNA fragments. Methods Enzymol. 1979;60:615–628. doi: 10.1016/s0076-6879(79)60058-7. [DOI] [PubMed] [Google Scholar]
  22. Weissbach H., Miller D. L., Hachmann J. Studies on the role of factor Ts in polypeptide synthesis. Arch Biochem Biophys. 1970 Mar;137(1):262–269. doi: 10.1016/0003-9861(70)90433-9. [DOI] [PubMed] [Google Scholar]
  23. Wittinghofer A., Frank R., Gast W. H., Leberman R. Polyphenylalanine synthesis by crystallized trypsin-modified EF-Tu.GDP. J Mol Biol. 1979 Aug 5;132(2):253–256. doi: 10.1016/0022-2836(79)90394-2. [DOI] [PubMed] [Google Scholar]
  24. Wittinghofer A., Frank R., Leberman R. Composition and properties of trypsin-cleaved elongation factor Tu. Eur J Biochem. 1980 Jul;108(2):423–431. doi: 10.1111/j.1432-1033.1980.tb04738.x. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES