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Analysis of Mutations in the 23S rRNA

  • Urmas Saarma
  • Birgit T. U. Lewicki
  • Tõnu Margus
  • Sulo Nigul
  • Jaanus Remme

Abstract

Several experimental approaches have been employed to study the structure and function of ribosomes (reviewed in Dahlberg, 1989, and Leclerc and Brakier-Gingras, 1990). One such approach is that of site-directed mutagenesis of the rRNA, most commonly in the rRNA of the bacterium Escherichia coli. This organism has seven rRNA operons and thus separation of the host encoded rRNA from that of the mutated plasmid-encoded rRNA is necessary for further biochemical studies. The problem can be overcome by using in vitro synthesized rRNA for reconstitution of the subunit. This has been sucessfully achieved for total reconstitution of functional 30S particles. In contrast, transcribed and unmodified 23S rRNA cannot form active particles in reconstitution assays. Therefore, 23S rRNA should be expressed in vivo followed by isolation of the ribosomes. It is possible to discriminate between activities of mutant and wild-type ribsomes by using a second mutation in the gene of 23S rRNA. One such mutation is the A to T transversion corresponding to the position 1067 of 23S rRNA which confers resistance to thiostrepton during cell-free translation (Thompson et al., 1988). Thiostrepton has a high affinity to wild-type ribosomes. U1067 ribosomes bind the drug with a reduced affinity (Thompson and Cundliffe, 1991). Therefore, A to U substitution at position 1067 can be used for selective inactivation of wild-type ribosomes and for physical separation of mutant ribosomes. This experimental approach has been used to analyze functional properties of mutations corresponding to positions G2505 and G2583 of 23S rRNA (Saarma and Remme, 1992). The most interesting finding was that mutations at position 2583 increased the translational accuracy. These results, together with those of mutations at positions G2505, G2583, G2607, and G2608 in vitro and G2505 and G2583 in vivo will be summarized here.

Keywords

Peptide Bond Formation Translational Accuracy Mutant Ribosome Thiostrepton Resistance Transcription Initiation Rate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Chamberlin, M., and Ring, J.; 1973; J. Biol. Chem. 248:2235PubMedGoogle Scholar
  2. Cunningham, P. R., Richard, R. B., Weitzmann, C. J., Nurse, K., and Ofengand, J.; 1991; Biochimie 73:789PubMedCrossRefGoogle Scholar
  3. Dahlberg, A.; 1989; Cell, 57:525PubMedCrossRefGoogle Scholar
  4. Gourse, R. L., Stark, M. J.R., and Dahlberg, A.;1983;Cell, 32:1347PubMedCrossRefGoogle Scholar
  5. Hausner, T.-P., Geigenmüller, U., and Nierhaus, K. J.; 1988; J. Biol. Chem. 263: 13103PubMedGoogle Scholar
  6. Krzyzosiak, W., Denman, R., Nurse, K., Hellmann, M., Boublik, M., Gehrke, C. W., Agris, P. F., and Ofengand, J.; 1987; Biochemistry 26:2353PubMedCrossRefGoogle Scholar
  7. Leclerc, D. and Brakier-Gingras, L.; 1990; Biochem. Cell Biol. 68:169PubMedCrossRefGoogle Scholar
  8. Lewicki, B. T. U., Margus, T., Remme, J., and Nierhaus, K. H.; 1993; J. Mol. Biol., in press.Google Scholar
  9. Melançon, P., Gravel, M., Boileau, G., and Brakier-Gingras, L.; 1987; Biochem. Cell Biol. 65:1022PubMedCrossRefGoogle Scholar
  10. Nierhaus, K. H.; 1991; Biochimie 73:739PubMedCrossRefGoogle Scholar
  11. Pestka, S.; 1977; In: “Molecular Mechanisms of Protein Biosynthesis”, Weisbach, H., Pestka, S. eds. Academic Press. New-York, San-Francisco, LondonGoogle Scholar
  12. Petrullo, L. A., Gallagher, P. J., and Elseviers, D.; 1983; Mol. Gen. Genet. 190:289PubMedCrossRefGoogle Scholar
  13. Prescott, C. D., and Kornau, H.-C.; 1992; Nucl. Acids Res. 20:1567PubMedCrossRefGoogle Scholar
  14. Rheinberger, H.-J., and Nierhaus, K. H.; 1990; Eur. J. Biochem. 193:643PubMedCrossRefGoogle Scholar
  15. Saarma, U., and Remme, J.; 1992; Nucl. Acids Res. 20:3147PubMedCrossRefGoogle Scholar
  16. Thompson, J., and Cundliffe, E.; 1991; Biochimie 73:1131PubMedCrossRefGoogle Scholar
  17. Thompson, J., Cundliffe, E., and Dahlberg, A.; 1988; J.Mol. Biol. 203:457PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Urmas Saarma
    • 1
  • Birgit T. U. Lewicki
    • 2
  • Tõnu Margus
    • 3
  • Sulo Nigul
    • 1
  • Jaanus Remme
    • 3
  1. 1.Estonian BiocentreEstonia
  2. 2.Max-Planck-Inst. f. Molekulare GenetikBerlin 33Germany
  3. 3.lnst. Chem. Phys. & BiophysEstonia

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