Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Oct 25;16(20):9631–9639. doi: 10.1093/nar/16.20.9631

A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

P Melançon 1, C Lemieux 1, L Brakier-Gingras 1
PMCID: PMC338768  PMID: 3054810

Abstract

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.

Full text

PDF
9631

Images in this article

9635Image
on p.9635

Selected References

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

  1. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  2. Brakier-Gingras L., Phoenix P. The control of accuracy during protein synthesis in Escherichia coli and perturbations of this control by streptomycin, neomycin, or ribosomal mutations. Can J Biochem Cell Biol. 1984 May;62(5):231–244. doi: 10.1139/o84-033. [DOI] [PubMed] [Google Scholar]
  3. Brosius J., Ullrich A., Raker M. A., Gray A., Dull T. J., Gutell R. R., Noller H. F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid. 1981 Jul;6(1):112–118. doi: 10.1016/0147-619x(81)90058-5. [DOI] [PubMed] [Google Scholar]
  4. Campuzano S., Cabañas M. J., Modolell J. The binding of non-cognate Tyr-tRNATyr to poly(uridylic acid)-programmed Escherichia coli ribosomes. Eur J Biochem. 1979 Oct;100(1):133–139. doi: 10.1111/j.1432-1033.1979.tb02041.x. [DOI] [PubMed] [Google Scholar]
  5. Chang F. N., Flaks J. G. Binding of dihydrostreptomycin to Escherichia coli ribosomes: characteristics and equilibrium of the reaction. Antimicrob Agents Chemother. 1972 Oct;2(4):294–307. doi: 10.1128/aac.2.4.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cundliffe E. On the nature of antibiotic binding sites in ribosomes. Biochimie. 1987 Aug;69(8):863–869. doi: 10.1016/0300-9084(87)90213-6. [DOI] [PubMed] [Google Scholar]
  8. Davis B. D., Chen L. L., Tai P. C. Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6164–6168. doi: 10.1073/pnas.83.16.6164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Girshovich A. S., Bochkareva E. S., Vasiliev V. D. Localization of elongation factor Tu on the ribosome. FEBS Lett. 1986 Mar 3;197(1-2):192–198. doi: 10.1016/0014-5793(86)80325-8. [DOI] [PubMed] [Google Scholar]
  10. Gornicki P., Nurse K., Hellmann W., Boublik M., Ofengand J. High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit. J Biol Chem. 1984 Aug 25;259(16):10493–10498. [PubMed] [Google Scholar]
  11. Gravel M., Melançon P., Brakier-Gingras L. Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli. Biochemistry. 1987 Sep 22;26(19):6227–6232. doi: 10.1021/bi00393a041. [DOI] [PubMed] [Google Scholar]
  12. Grisé-Miron L., Noreau J., Melançon P., Brakier-Gingras L. Comparison of the misreading induced by streptomycin and neomycin. Biochim Biophys Acta. 1981 Nov 27;656(1):103–110. doi: 10.1016/0005-2787(81)90032-0. [DOI] [PubMed] [Google Scholar]
  13. Hanas J. S., Simpson M. V. Fluorescence studies on a streptomycin-induced conformational change in ribosomes which correlates with misreading. J Biol Chem. 1986 May 25;261(15):6670–6676. [PubMed] [Google Scholar]
  14. Jelenc P. C., Kurland C. G. Multiple effects of kanamycin on translational accuracy. Mol Gen Genet. 1984;194(1-2):195–199. doi: 10.1007/BF00383516. [DOI] [PubMed] [Google Scholar]
  15. Lake J. A. Aminoacyl-tRNA binding at the recognition site is the first step of the elongation cycle of protein synthesis. Proc Natl Acad Sci U S A. 1977 May;74(5):1903–1907. doi: 10.1073/pnas.74.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lake J. A. Evolving ribosome structure: domains in archaebacteria, eubacteria, eocytes and eukaryotes. Annu Rev Biochem. 1985;54:507–530. doi: 10.1146/annurev.bi.54.070185.002451. [DOI] [PubMed] [Google Scholar]
  17. Lando D., Cousin M. A., Ojasoo T., Raymond J. P. Paromomycin and dihydrostreptomycin binding to Escherichia coli ribosomes. Eur J Biochem. 1976 Jul 15;66(3):597–606. doi: 10.1111/j.1432-1033.1976.tb10587.x. [DOI] [PubMed] [Google Scholar]
  18. Langer J. A., Lake J. A. Elongation factor Tu localized on the exterior surface of the small ribosomal subunit. J Mol Biol. 1986 Feb 20;187(4):617–621. doi: 10.1016/0022-2836(86)90339-6. [DOI] [PubMed] [Google Scholar]
  19. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Moazed D., Noller H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987 Jun 4;327(6121):389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
  22. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  23. Montandon P. E., Nicolas P., Schürmann P., Stutz E. Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position. Nucleic Acids Res. 1985 Jun 25;13(12):4299–4310. doi: 10.1093/nar/13.12.4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Montandon P. E., Wagner R., Stutz E. E. coli ribosomes with a C912 to U base change in the 16S rRNA are streptomycin resistant. EMBO J. 1986 Dec 20;5(13):3705–3708. doi: 10.1002/j.1460-2075.1986.tb04703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Noreau J., Grisé-Miron L., Brakier-Gingras L. Comparison of the action of streptomycin and neomycin on the structure of the bacterial ribosome. Biochim Biophys Acta. 1980 Jun 27;608(1):72–81. doi: 10.1016/0005-2787(80)90135-5. [DOI] [PubMed] [Google Scholar]
  26. Oakes M. I., Clark M. W., Henderson E., Lake J. A. DNA hybridization electron microscopy: ribosomal RNA nucleotides 1392-1407 are exposed in the cleft of the small subunit. Proc Natl Acad Sci U S A. 1986 Jan;83(2):275–279. doi: 10.1073/pnas.83.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ruusala T., Kurland C. G. Streptomycin preferentially perturbs ribosomal proofreading. Mol Gen Genet. 1984;198(2):100–104. doi: 10.1007/BF00328707. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sigmund C. D., Morgan E. A. Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5602–5606. doi: 10.1073/pnas.79.18.5602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smailov S. K., Gavrilova L. P. Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. FEBS Lett. 1985 Nov 11;192(1):165–169. doi: 10.1016/0014-5793(85)80065-x. [DOI] [PubMed] [Google Scholar]
  31. Stöffler G., Stöffler-Meilicke M. Immunoelectron microscopy of ribosomes. Annu Rev Biophys Bioeng. 1984;13:303–330. doi: 10.1146/annurev.bb.13.060184.001511. [DOI] [PubMed] [Google Scholar]
  32. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thompson R. C., Dix D. B., Gerson R. B., Karim A. M. Effect of Mg2+ concentration, polyamines, streptomycin, and mutations in ribosomal proteins on the accuracy of the two-step selection of aminoacyl-tRNAs in protein biosynthesis. J Biol Chem. 1981 Jul 10;256(13):6676–6681. [PubMed] [Google Scholar]
  35. Thompson R. C. EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem Sci. 1988 Mar;13(3):91–93. doi: 10.1016/0968-0004(88)90047-3. [DOI] [PubMed] [Google Scholar]
  36. Trempe M. R., Ohgi K., Glitz D. G. Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy. J Biol Chem. 1982 Aug 25;257(16):9822–9829. [PubMed] [Google Scholar]

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

RESOURCES