Molecular and General Genetics MGG

, Volume 198, Issue 1, pp 90–99

Kinetic impairment of restrictive streptomycin-resistant ribosomes

  • K. Bohman
  • T. Ruusala
  • P. C. Jelenc
  • C. G. Kurland
Article

Summary

Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation.

A complementary pattern is seen in vitro, where ribosomes from the restrictive Smr bacteria translate poly(U) with a significantly lower missense error frequency than wild-type ribosomes, and also show an increased Michaelis constant (KM) with respect to their substrate, i.e. ternary complexes. Both effects are correlated with the more aggressive proofreading function that is characteristic of these restrictive ribosomes. In contrast, ribosomes isolated from the non-restrictive Smr bacteria do not show any major difference in either proofreading or missense error in vitro when compared to the wild type.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andersson DI, Kurland CG (1983) Ram ribosomes are defective proofreaders. Mol Gen Genet 191:378–381Google Scholar
  2. Andersson DI, Bohman K, Isaksson LA, Kurland CG (1982) Translation rates and misreading characteristics of rpsD mutants in Escherichia coli. Mol Gen Genet 187:467–472Google Scholar
  3. Arai KJ, Kawakita M, Kaziro Y (1972) Studies on polypeptide elongation factors from Escherichia coli. J Biol Chem 247:7029–7037Google Scholar
  4. Birge EA, Kurland CG (1969) Altered ribosomal protein in streptomycin dependent Escherichia coli. Science 166:1282–1284Google Scholar
  5. Bouadloun F, Donner D, Kurland CG (1983) Codon-specific missense errors in vivo. EMBO J 2:1351–1356Google Scholar
  6. Breckenridge L, Gorini L (1970) Genetic analysis of streptomycin resistance in Escherichia coli. Genetics 65:9–25Google Scholar
  7. Couturier M, Desmet L, Thomas R (1964) High pleiotropy of streptomycin mutations in Escherichia coli. Biochem Biophys Res Commun 16:244–248Google Scholar
  8. Ehrenberg M, Kurland CG (1984) Costs of accuracy determined by a maximal growth rate constraint. Q Rev Biophys 17:45–82Google Scholar
  9. Fersht A (1977) Enzyme structure and mechanism. WH Freeman and Co, Reading, San FranciscoGoogle Scholar
  10. Flaks JG, Cox EC, White JR (1962) Inhibition of polypeptide synthesis by streptomycin. Biochem Biophys Res Commun 7:385–389Google Scholar
  11. Funatsu G, Wittmann HG (1972) Ribosomal proteins XXXIII. Location of aminoacid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin. J Mol Biol 68:547–550Google Scholar
  12. Galas DJ, Branscomb EW (1976) Ribosome slowed by mutation to streptomycin resistance. Nature 262:617–619Google Scholar
  13. Gorini L (1971) Ribosomal discrimination of tRNAs. Nature New Biol 234:261–264Google Scholar
  14. Gorini L, Jacoby AG, Breckenridge L (1966) Ribosomal ambiguity. Cold Spring Harbor Symp Quant Biol 31:657–664Google Scholar
  15. Hopfield JJ (1974) Kinetic proofreading: a new mechnism for reducing errors in biosynthetic processes requiring high specificity. Proc Natl Acad Sci USA 71:4135–4139Google Scholar
  16. Isaksson LA, Sköld SE, Skjöldebrand J, Takata R (1977) A procedure for isolation of spontaneous mutants with temperature sensitive synthesis of RNA and/or protein. Mol Gen Genet 156:233–237Google Scholar
  17. Jelenc PC (1980) Rapid purification of highly active ribosomes from Escherichia coli. Anal Biochem 105:369–374Google Scholar
  18. Jelenc PC, Kurland CG (1979) Nucleoside triphosphate regeneration decreases the frequency of translation errors. Proc Natl Acad Sci USA 76:3174–3178Google Scholar
  19. Jelenc PC, Kurland CG (1984) Multiple effects of kanamycin on translational accuracy. Mol Gen Genet 194:195–199Google Scholar
  20. Kalnins A, Otto K, Rüther U, Müller-Hill B (1983) Sequence of the lacZ gene of Escherichia coli. EMBO J 2:593–597Google Scholar
  21. Kurland CG, Ehrenberg M (1984) Optimization of translational accuracy. Prog Nucl Acid Res Mol Biol (in press)Google Scholar
  22. Lebermann R, Antonsson B, Giovanelli R, Guariguata R, Schumann R, Wittinghofer A (1980) A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu. Anal Biochem 104:29–36Google Scholar
  23. Lederberg EM, Cavalli-Sforza LL, Lederberg J (1964) Interaction of streptomycin and a suppressor for galactose fermentation in E. coli K-12. Proc Natl Acad Sci USA 51:678–681Google Scholar
  24. Miller CP, Bohnhoff M (1947) Two streptomycin-resistant variants of meningococcus. J Bacteriol 54:467–475Google Scholar
  25. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  26. Miller JH, Coulondre C, Farabaugh PJ (1978) Correlation of nonsense sites in the lacI gene with specific codons in the nucleotide sequence. Nature 274:770–775Google Scholar
  27. Morris JG (1974) A biologist's physical chemistry. Edward Arnold Ltd, London, p 287Google Scholar
  28. Müller-Hill B, Kania J (1974) Lac repressor can be fused to β-galactosidase. Nature 249:561–563Google Scholar
  29. Newcombe HB, Hawirko R (1949) Spontaneous mutation to streptomycin resistance and dependence in Escherichia coli. J Bacteriol 56:565–571Google Scholar
  30. Ninio J (1974) A semi-quantiative treatment of missense and nonsense suppression in the strA and ram ribosomal mutants of Escherichia coli. Evaluation of some molecular parameters of translation in vivo. J Mol Biol 84:297–313Google Scholar
  31. Ninio J (1975) Kinetic amplification of enzyme discrimination. Biochimie 57:587–595Google Scholar
  32. Olsson M, Isaksson LA (1979) Analysis of rpsD mutations in Escherichia coli I Comparision of mutants with various alterations in ribosomal protein S4. Mol Gen Genet 169:251–257Google Scholar
  33. Ozaki M, Mizushima S, Nomura M (1969) Identification and functional characterization of the protein controlled by the streptomycin resistant locus in E. coli. Nature 222:333–339Google Scholar
  34. Pedersen S (1984) In Escherichia coli individual genes are translated with different rates in vivo. Alfred Benzon Symposium 19, Munksgaard, Copenhagen (in press)Google Scholar
  35. Piepersberg W, Noseda V, Böck A (1979) Bacterial ribosomes with two ambiquity mutations: Effects on translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis. Mol Gen Genet 171:23–34Google Scholar
  36. Ruusala T, Ehrenberg M, Kurland CG (1982a) Catalytic effects of elongation factor Ts on polypeptide synthesis. EMBO J 1:75–78Google Scholar
  37. Ruusala T, Ehrenberg M, Kurland CG (1982b) Is there proofreading during polypeptide synthesis? EMBO J 1:741–745Google Scholar
  38. Ruusala T, Andersson DI, Ehrenberg M, Kurland CG (1984) Hyper-accurate ribosomes inhibit growth. EMBO J (In press)Google Scholar
  39. Schleif R, Hess W, Finkelstein S, Ellis D (1973) Induction kinetics of the L-arabinose operon of Escherichia coli. J Bacteriol 115:9–14Google Scholar
  40. Speyer JF, Lengyel P, Basilio C (1962) Ribosomal localization of streptomycin sensitivity. Proc Natl Acad Sci USA 48:684–686Google Scholar
  41. Spotts CR, Stanier RY (1961) Mechanism of streptomycin action on bacteria: A unitary hypothesis. Nature 192:633–637Google Scholar
  42. Thompson RC, Dix DB, Gerson RB, Karim AM (1981) 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 256:6676–6681Google Scholar
  43. Wagner EGH, Kurland CG (1980) Escherichia coli elongation factor G blocks stringent factor. Biochemistry 19:1234–1240Google Scholar
  44. Wagner EGH, Jelenc PC, Ehrenberg M, Kurland CG (1982) Rate of elongation of polyphenylalanine in vitro. Eur J Biochem 122:193–197Google Scholar
  45. Yates JL (1979) Role of ribosomal protein S12 in discrimination of aminoacyl-tRNA. J Biol Chem 254:11550–11554Google Scholar
  46. Zengel JM, Young R, Dennis PP, Nomura M (1977) Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotrophic, streptomycin-resistant mutants of Escherichia coli. J Bacteriol 129:1326–1329Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • K. Bohman
    • 1
  • T. Ruusala
    • 1
  • P. C. Jelenc
    • 1
  • C. G. Kurland
    • 1
  1. 1.Department of Molecular Biology, BiomedicumUppsalaSweden

Personalised recommendations