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. 1996 Jun;178(11):3246–3251. doi: 10.1128/jb.178.11.3246-3251.1996

Tet(M)-promoted release of tetracycline from ribosomes is GTP dependent.

V Burdett 1
PMCID: PMC178077  PMID: 8655505

Abstract

Tet(M) protein, which displays homology to elongation factor G (EF-G), interacts with the protein biosynthetic machinery to render this process resistant to tetracycline in vivo and in vitro. To clarify the basis of the resistance mechanism, the effects of Tet(M) on several reactions which occur during protein synthesis were examined. The mechanism of action of Tet(M) has been clarified by two observations. The protein relieves tetracycline inhibition of factor-dependent tRNA binding and dramatically reduces the affinity of ribosomes for tetracycline when GTP is present. This reduction in drug affinity appears to be due to a large increase in the rate of tetracycline dissociation. Addition of Tet(M) to ribosome-tetracycline complexes results in displacement of bound drug. And, while Tet(M) and EF-G GTPase activities are tetracycline resistant, the two proteins differ in their sensitivities to fusidic acid, with the latter activity inhibited by the drug. Furthermore, while Tet(M) protects translation from tetracycline inhibition in a defined system, it is unable to substitute for either EF-G or elongation factor Tu.

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Selected References

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  1. Axelson J. T., Bodley J. W., Chen J. Y., Dunlop P. C., Rosenthal L. P., Viskup R. W., Walseth T. F. Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment. Anal Biochem. 1984 Nov 1;142(2):373–377. doi: 10.1016/0003-2697(84)90479-2. [DOI] [PubMed] [Google Scholar]
  2. Burdett V. Nucleotide sequence of the tet(M) gene of Tn916. Nucleic Acids Res. 1990 Oct 25;18(20):6137–6137. doi: 10.1093/nar/18.20.6137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burdett V. Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline. J Biol Chem. 1991 Feb 15;266(5):2872–2877. [PubMed] [Google Scholar]
  4. Burdett V. Streptococcal tetracycline resistance mediated at the level of protein synthesis. J Bacteriol. 1986 Feb;165(2):564–569. doi: 10.1128/jb.165.2.564-569.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Charpentier E., Gerbaud G., Courvalin P. Characterization of a new class of tetracycline-resistance gene tet(S) in Listeria monocytogenes BM4210. Gene. 1993 Sep 6;131(1):27–34. doi: 10.1016/0378-1119(93)90665-p. [DOI] [PubMed] [Google Scholar]
  7. Cunningham P. R., Nurse K., Bakin A., Weitzmann C. J., Pflumm M., Ofengand J. Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function. Biochemistry. 1992 Dec 8;31(48):12012–12022. doi: 10.1021/bi00163a008. [DOI] [PubMed] [Google Scholar]
  8. Ducruix A., Hounwanou N., Reinbolt J., Boulanger Y., Blanquet S. Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase. Biochim Biophys Acta. 1983 Nov 17;741(2):244–250. doi: 10.1016/0167-4781(83)90065-9. [DOI] [PubMed] [Google Scholar]
  9. Epe B., Woolley P. The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy. EMBO J. 1984 Jan;3(1):121–126. doi: 10.1002/j.1460-2075.1984.tb01771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grewal J., Manavathu E. K., Taylor D. E. Effect of mutational alteration of Asn-128 in the putative GTP-binding domain of tetracycline resistance determinant Tet(O) from Campylobacter jejuni. Antimicrob Agents Chemother. 1993 Dec;37(12):2645–2649. doi: 10.1128/aac.37.12.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hamel E., Koka M., Nakamoto T. Requirement of an Escherichia coli 50 S ribosomal protein component for effective interaction of the ribosome with T and G factors and with guanosine triphosphate. J Biol Chem. 1972 Feb 10;247(3):805–814. [PubMed] [Google Scholar]
  12. Johanson U., Hughes D. Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium. Gene. 1994 May 27;143(1):55–59. doi: 10.1016/0378-1119(94)90604-1. [DOI] [PubMed] [Google Scholar]
  13. Knudsen C. R., Clark B. F., Degn B., Wiborg O. One-step purification of E. coli elongation factor Tu. Biochem Int. 1992 Oct;28(2):353–362. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Manavathu E. K., Fernandez C. L., Cooperman B. S., Taylor D. E. Molecular studies on the mechanism of tetracycline resistance mediated by Tet(O). Antimicrob Agents Chemother. 1990 Jan;34(1):71–77. doi: 10.1128/aac.34.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mesters J. R., Potapov A. P., de Graaf J. M., Kraal B. Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations. J Mol Biol. 1994 Oct 7;242(5):644–654. doi: 10.1006/jmbi.1994.1614. [DOI] [PubMed] [Google Scholar]
  17. Nikolich M. P., Shoemaker N. B., Salyers A. A. A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance. Antimicrob Agents Chemother. 1992 May;36(5):1005–1012. doi: 10.1128/aac.36.5.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohnuki T., Katoh T., Imanaka T., Aiba S. Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes. J Bacteriol. 1985 Mar;161(3):1010–1016. doi: 10.1128/jb.161.3.1010-1016.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peterson E. T., Uhlenbeck O. C. Determination of recognition nucleotides for Escherichia coli phenylalanyl-tRNA synthetase. Biochemistry. 1992 Oct 27;31(42):10380–10389. doi: 10.1021/bi00157a028. [DOI] [PubMed] [Google Scholar]
  20. Rheinberger H. J., Nierhaus K. H. Allosteric interactions between the ribosomal transfer RNA-binding sites A and E. J Biol Chem. 1986 Jul 15;261(20):9133–9139. [PubMed] [Google Scholar]
  21. Sloan J., McMurry L. M., Lyras D., Levy S. B., Rood J. I. The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants. Mol Microbiol. 1994 Jan;11(2):403–415. doi: 10.1111/j.1365-2958.1994.tb00320.x. [DOI] [PubMed] [Google Scholar]
  22. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Voigt J., Nagel K. Regulation of elongation factor G GTPase activity by the ribosomal state. The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA. J Biol Chem. 1993 Jan 5;268(1):100–106. [PubMed] [Google Scholar]
  24. Willie G. R., Richman N., Godtfredsen W. P., Bodley J. W. Some characteristics of and structural requirements for the interaction of 24,25-dihydrofusidic acid with ribosome - elongation factor g Complexes. Biochemistry. 1975 Apr 22;14(8):1713–1718. doi: 10.1021/bi00679a025. [DOI] [PubMed] [Google Scholar]

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