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Molecular and General Genetics MGG

, Volume 191, Issue 2, pp 207–212 | Cite as

The frequency of transcriptional and translational errors at nonsense codons in the lacZ gene of Escherichia coli

  • R. F. Rosenberger
  • J. Hilton
Article

Summary

Nonsense alleles in the lacZ gene of E. coli do not completely eliminate enzyme activity as errors during protein synthesis allow some chains to be completed. The relative contributions of transcriptional and translational errors to this leakiness were investigated by two methods: the introduction of rho alleles into extreme-polar mutants and the kinetics of β-galactosidase induction. Virtually all the errors appeared to be transcriptional in the case of two extreme-polar and one non-polar mutation. These alleles should prove useful for further in vivo investigations of RNA polymerase accuracy. With two other non-polar alleles, transcriptional mistakes were low and translational ones high. The frequency of RNA polymerase errors was context-dependent and varied for different nonsense codons in the same position and for the same codon in different positions. The reasons why some alleles showed no activity due to translational errors could not be clearly established. However, increasing the rates of ribosomal errors from one such allele with streptomycin raised the contribution of ribosomal errors to activity markedly and non-linearly. Translational mistakes may give rise to active enzyme only if the monomers are formed at a rate sufficient for effective aggregation to the normal tetramer.

Keywords

Enzyme Active Enzyme Codon Protein Synthesis Streptomycin 
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. Alpers DH, Appel SH, Tomkins GM (1965) A spectrophotometric assay for thioglactoside transacetylase. J Biol Chem 240:10–13Google 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. Bouadloun F, Kurland CG (1983) Codon-specific missense errors in vitro. EMBO J (in press)Google Scholar
  4. Brake AJ, Fowler AV, Zabin T, Kassia J, Muller-Hill B (1978) β-galactosidase chimeras: Primary structure of a lac repressor β-galactosidase protein. Proc Nat Acad Sci USA 75:4824–4827Google Scholar
  5. Carter T, Newton A (1971) New polarity suppressors in Escherichia coli: suppression and messenger RNA stability. Proc Nat Acad Sci USA 68:2962–2966Google Scholar
  6. Dabbs ER, Looman K (1981) An antibiotic-dependant conditional lethal mutant with a lesion affecting transcription and translation. Mol Gen Genet 184:224–229Google Scholar
  7. Davis BD, Mingioli ES (1950) Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol 60:17–28Google Scholar
  8. Edelmann P, Gallant J (1977) On the translational theory of aging. Proc Nat Acad Sci USA 74:3396–3398Google Scholar
  9. Fowler AV, Zabin I (1978) Amino acid sequence of β-galactosidase. XI. Peptide ordering procedures and the complete sequence. J. Biol. Chem, pp 5521–5525Google Scholar
  10. Friedman DI, Schaner AT, Baumann MR, Baron LS, Adhya SL (1981) Evidence that ribosomal protein S10 participates in control of transcription termination. Proc Nat Acad Sci USA 78:1115–1118Google Scholar
  11. Gallant J, Palmer (1979) Error propagation in viable cells. Mech Ageing Dev 10:27–38Google Scholar
  12. Gallant J, Foley D (1980) On the causes and prevention of mistranslation. In: Chambliss G, Craven CR, Davis T, Kahan L, Nomura M (eds) Ribosomes: Structure, function and genetics. University Park Press, Baltimore, pp 615–640Google Scholar
  13. Gallant J, Erlich H, Weiss R, Palmer L, Nyari L (1982) Nonsense suppression in aminoacyl-t-RNA limited cells. Mol Gen Genet 186:221–227Google Scholar
  14. Gorini L (1971) Ribosomal discrimination of tRNAs. Nature (London) New Biol 234:261–264Google Scholar
  15. Gorini L (1974) Streptomycin and misreading of the genetic code. In: Nomura M, Tissieres A, Lengyell P (eds) Ribosomes. Cold Spring Harbor Laboratory, New York, pp 791–803Google Scholar
  16. Kelley RL, Yanofsky C (1982) trp aporepressor production is controlled by autogenous regulation and inefficient translation. Proc Nat Acad Sci USA 79:3120–3124Google Scholar
  17. Kogut M, Lightbown JW, Isaacson P (1965) Effects of dihydrostreptomycin treatment on the growth of Escherichia coli after removal of extracellular antibiotic. J Gen Microbiol 39:165–183Google Scholar
  18. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  19. Morse DE, Guertin M (1972) Amber SuA mutations which relieve polarity. J Mol Biol 63:605–608Google Scholar
  20. O'Farrell P (1978) The suppression of defective translation by ppGpp and its role in the stringent response. Cell 14:545–557Google Scholar
  21. Parker T, Friesen JD (1980) Two out of three codon reading leading to mistranslation in vivo. Mol Gen Genet 177:439–445Google Scholar
  22. Parker J, Johnson TC, Borgia PT (1980) Mistranslation in cells infected with the bacteriophate MS2: direct evidence of Lys for Asn substitution. Mol Gen Genet 180:275–281Google Scholar
  23. Piepersberg W, Böck A, Wittmann HG (1975) Effect, of different mutations in ribosomal protein S5 of Escherichia coli on translational fidelity. Mol Gen Genet 140:91–100Google Scholar
  24. Piepersberg W, Noseda V, Böck A (1979) Bacterial ribosomes with two ambiguity mutations: effects on translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis. Mol Gen Genet 171:23–34Google Scholar
  25. Read SM, Northcote DH (1981) Minimisation of variation in the response to different proteins of the Coomassie Blue G dyebinding assay for protein. Anal Biochem 116:53–64Google Scholar
  26. Richardson JP, Grimley C, Lowery C (1975) Transcription termination factor rho activity is altered in E. coli with SuA mutations. Proc Nat Acad Sci USA 72:1725–1728Google Scholar
  27. Rosenberger RF, Foskett G (1981) An estimate of the frequency of in vivo transcriptional errors at a nonsense codon in E. coli. Mol Gen Genet 183:561–563Google Scholar
  28. Rosner JL (1972) Formation, induction and curing of P1 lysogens. Virology 48:679–689Google Scholar
  29. Sarthy A, Fowler A, Zabin I, Beckwith J (1979) Use of a gene fusion to determine a partial sequence of alkaline phosphatase. J Bacteriol 139:932–939Google Scholar
  30. Schlessinger D, Medoff G (1975) Streptomycin, dihydrostreptomycin and the gentamycins. In: Corcoran JW, Hahn FE (eds) Antibiotics, vol III. Springer, Berlin Heidelberg New York, pp 535–550Google Scholar
  31. Ullman A, Monod J (1969) On the effect of divalent ions and protein concentration upon the renaturation of β-galactosidase from E. coli. Biochem Biophys Res Commun 35:35–42Google Scholar
  32. Welpy TK, Fowler AV, Beckwith JR, Zabin I (1980) Position of early nonsense and deletion mutations in lacZ. J Bacteriol 142:732–734Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • R. F. Rosenberger
    • 1
  • J. Hilton
    • 1
  1. 1.Genetics DivisionThe Natonal Institute for Medical ResearchLondonEngland

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