1887

Abstract

An epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu of was inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 of suggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 of By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.

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/content/journal/micro/10.1099/00221287-144-8-2241
1998-08-01
2024-04-27
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References

  1. Appel J.R., Pinilla C., Niman H., Houghten R. 1990; Elucidation of discontinuous linear determinants in peptides.. J Immunol 144:976–983
    [Google Scholar]
  2. Baldauf S.L., Palmer J.D., Doolittle W.F. 1996; The root of the universal tree and the origin of eukaryotes based on elongation factor phylogeny. . Proc Natl Acad Sci USA 93:7749–7754
    [Google Scholar]
  3. Berchtold H., Reshetnikova L., Reiser C.O.A., Schirmer N.K., Sprinzl M., Hilgenfeld R. 1993; Crystal structure of active elongation factor Tu reveals major domain rearrangements. . Nature 365:126–132
    [Google Scholar]
  4. Filer D., Furano A.V. 1980; Portions of the gene encoding elongation factor Tu are highly conserved. . J Biol Chem 255:728–734
    [Google Scholar]
  5. Filer D., Furano A.V. 1981a; The conservation of DNA sequences over very long periods of evolutionary time. . Eur J Biochem 120:67–77
    [Google Scholar]
  6. Filer D., Furano A.V. 1981b; Duplication of thetuf gene, which encodes peptide chain elongation factor Tu, is widespread in gram-negative bacteria.. J Bacteriol 148:1006–1011
    [Google Scholar]
  7. Frank R. 1992; Spot-Synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.. Tetrahedron 48:9217–9232
    [Google Scholar]
  8. Frank R., Overwin H. 1996; SPOT Synthesis: epitope analysis with arrays of synthetic peptides prepared on cellulose membranes.. In Methods in Molecular Biology 66 Epitope Mapping Protocols pp. 149–169 Morris G.E. Edited by Totowa: Humana Press;
    [Google Scholar]
  9. Kjeldgaard M., Nyborg J. 1992; Refined structure of elongation factor EF-Tu fromEscherichia coli. . J Mol Biol 223:721–742
    [Google Scholar]
  10. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 227:680–685
    [Google Scholar]
  11. Ludwig W., Weizenegger M., Betzl D., Leidei E., Lenz T., Ludvigsen A., Möllenhoff D., Wenzig P., Schleiffer K.-H. 1990; Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: structural and phylogenetic evaluations.. Arch Microbiol 153:241–247
    [Google Scholar]
  12. Lüneberg E., Kamla V., Hadding U., Frosch M. 1991; Sequence and expression inEscherichia coli of aMycoplasma hominis gene encoding elongation factor Tu.. Gene 102:123–127
    [Google Scholar]
  13. Miller D.L., Weissbach H. 1977; Factors involved in the transfer of aminoacyl-tRNA to the ribosome.. In Molecular Mechanisms of Protein Biosynthesis pp. 323–374 Weissbach H., Pestka S. Edited by New York: Academic Press;
    [Google Scholar]
  14. Nissen P., Kjeldgard M., Thirup S., Polekhina G., Reshetnikova L., Clark B.F.C., Nyborg J. 1995; Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog.. Science 270:1464–1472
    [Google Scholar]
  15. Nock S., Grillenbeck N., Ahmadian M.R., Ribeiro S., Kreutzer R., Sprinzl M. 1995; Properties of isolated domains of the elongation factor Tu fromThermus thermophilus HB 8.. Eur J Biochem 234:132–139
    [Google Scholar]
  16. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  17. Schmitt K., Däubener W., Bitter-Suermann D., Hadding U. 1988; A safe and efficient method for elimination of cell mycoplasmas using ciprofloxacin.. J Immunol Methods 109:17–25
    [Google Scholar]
  18. Sela S., Yogev D., Razin S., Bercovier H. 1989; Duplication of thetuf gene: a new insight into the phylogeny of eubacteria.. J Bacteriol 171:581–584
    [Google Scholar]
  19. Weber S., Lottspeich F., Köhl J. 1995; An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains.. J Bacteriol 175:11–19
    [Google Scholar]
  20. Weijland A., Parmeggiani A. 1994; Why do two EF-Tu molecules act in the elongation cycle of protein biosynthesis?. Trends Biochem Sci 19:188–193
    [Google Scholar]
  21. Wenzig P., Schleifer K.-H. 1989; Unusually strong immunological cross-reaction between elongation factor Tu ofEscherichia coli andBacillus subtilis. . Arch Microbiol 152:258–262
    [Google Scholar]
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