Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Jan;82(2):483–487. doi: 10.1073/pnas.82.2.483

Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA.

R L Kelley, C Yanofsky
PMCID: PMC397063  PMID: 3881764

Abstract

Several classes of trp repressor mutants were selected and analyzed in vivo. Mutants that produced repressors with either enhanced or reduced activity were obtained. One class of mutants produced inactive or slightly active repressors that were trans-dominant to the wild-type repressor. The amino acid substitutions in many of these repressors were clustered in a segment of the polypeptide that is homologous to the DNA recognition domain of the lambda cro repressor. A second functionally important region of the trp repressor was identified; this region could participate in L-tryptophan binding. Observations with trpR nonsense mutants suggest that the first 67 residues of the repressor polypeptide are sufficient for subunit association.

Full text

PDF
483

Selected References

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

  1. Adler K., Beyreuther K., Fanning E., Geisler N., Gronenborn B., Klemm A., Müller-Hill B., Pfahl M., Schmitz A. How lac repressor binds to DNA. Nature. 1972 Jun 9;237(5354):322–327. doi: 10.1038/237322a0. [DOI] [PubMed] [Google Scholar]
  2. Anderson W. F., Ohlendorf D. H., Takeda Y., Matthews B. W. Structure of the cro repressor from bacteriophage lambda and its interaction with DNA. Nature. 1981 Apr 30;290(5809):754–758. doi: 10.1038/290754a0. [DOI] [PubMed] [Google Scholar]
  3. Anderson W. F. Proposed alpha-helical super-secondary structure associated with protein-dna recognition. J Mol Biol. 1982 Aug 25;159(4):745–751. doi: 10.1016/0022-2836(82)90111-5. [DOI] [PubMed] [Google Scholar]
  4. Bennett G. N., Yanofsky C. Sequence analysis of operator constitutive mutants of the tryptophan operon of Escherichia coli. J Mol Biol. 1978 May 15;121(2):179–192. doi: 10.1016/s0022-2836(78)80004-7. [DOI] [PubMed] [Google Scholar]
  5. Betz J. L., Sadler J. R. Tight-binding repressors of the lactose operon. J Mol Biol. 1976 Aug 5;105(2):293–319. doi: 10.1016/0022-2836(76)90113-3. [DOI] [PubMed] [Google Scholar]
  6. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown K. D. Regulation of aromatic amino acid biosynthesis Escherichia coli K12. Genetics. 1968 Sep;60(1):31–48. doi: 10.1093/genetics/60.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli. Nature. 1984 Sep 20;311(5983):232–235. doi: 10.1038/311232a0. [DOI] [PubMed] [Google Scholar]
  10. Gunsalus R. P., Yanofsky C. Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7117–7121. doi: 10.1073/pnas.77.12.7117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HENNING U., YANOFSKY C. Amino acid replacements associated with reversion and recombination within the A gene. Proc Natl Acad Sci U S A. 1962 Sep 15;48:1497–1504. doi: 10.1073/pnas.48.9.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hecht M. H., Nelson H. C., Sauer R. T. Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding. Proc Natl Acad Sci U S A. 1983 May;80(9):2676–2680. doi: 10.1073/pnas.80.9.2676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Joachimiak A., Kelley R. L., Gunsalus R. P., Yanofsky C., Sigler P. B. Purification and characterization of trp aporepressor. Proc Natl Acad Sci U S A. 1983 Feb;80(3):668–672. doi: 10.1073/pnas.80.3.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Joachimiak A., Schevitz R. W., Kelley R. L., Yanofsky C., Sigler P. B. Functional inferences from crystals of Escherichia coli trp repressor. J Biol Chem. 1983 Oct 25;258(20):12641–12643. [PubMed] [Google Scholar]
  15. Kelley R. L., Yanofsky C. Trp aporepressor production is controlled by autogenous regulation and inefficient translation. Proc Natl Acad Sci U S A. 1982 May;79(10):3120–3124. doi: 10.1073/pnas.79.10.3120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laughon A., Scott M. P. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins. Nature. 1984 Jul 5;310(5972):25–31. doi: 10.1038/310025a0. [DOI] [PubMed] [Google Scholar]
  17. Matthews B. W., Ohlendorf D. H., Anderson W. F., Fisher R. G., Takeda Y. Cro repressor protein and its interaction with DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):427–433. doi: 10.1101/sqb.1983.047.01.050. [DOI] [PubMed] [Google Scholar]
  18. Matthews B. W., Ohlendorf D. H., Anderson W. F., Takeda Y. Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1428–1432. doi: 10.1073/pnas.79.5.1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [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. Miller J. H., Coulondre C., Hofer M., Schmeissner U., Sommer H., Schmitz A., Lu P. Genetic studies of the lac repressor. IX. Generation of altered proteins by the suppression of nonsence mutations. J Mol Biol. 1979 Jun 25;131(2):191–222. doi: 10.1016/0022-2836(79)90073-1. [DOI] [PubMed] [Google Scholar]
  22. Miller J. H., Schmeissner U. Genetic studies of the lac repressor. X. Analysis of missense mutations in the lacI gene. J Mol Biol. 1979 Jun 25;131(2):223–248. doi: 10.1016/0022-2836(79)90074-3. [DOI] [PubMed] [Google Scholar]
  23. Nelson H. C., Hecht M. H., Sauer R. T. Mutations defining the operator-binding sites of bacteriophage lambda repressor. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):441–449. doi: 10.1101/sqb.1983.047.01.052. [DOI] [PubMed] [Google Scholar]
  24. Ogata R. T., Gilbert W. An amino-terminal fragment of lac repressor binds specifically to lac operator. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5851–5854. doi: 10.1073/pnas.75.12.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ohlendorf D. H., Anderson W. F., Fisher R. G., Takeda Y., Matthews B. W. The molecular basis of DNA-protein recognition inferred from the structure of cro repressor. Nature. 1982 Aug 19;298(5876):718–723. doi: 10.1038/298718a0. [DOI] [PubMed] [Google Scholar]
  26. Ohlendorf D. H., Anderson W. F., Matthews B. W. Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor. J Mol Evol. 1983;19(2):109–114. doi: 10.1007/BF02300748. [DOI] [PubMed] [Google Scholar]
  27. Pabo C. O., Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. doi: 10.1038/298443a0. [DOI] [PubMed] [Google Scholar]
  28. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  29. Pabo C. O., Sauer R. T., Sturtevant J. M., Ptashne M. The lambda repressor contains two domains. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1608–1612. doi: 10.1073/pnas.76.4.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pfahl M. Characteristics of tight binding repressors of the lac operon. J Mol Biol. 1981 Mar 25;147(1):1–10. doi: 10.1016/0022-2836(81)90075-9. [DOI] [PubMed] [Google Scholar]
  31. Pfahl M. lac Repressor-operator interaction. Analysis of the X86 repressor mutant. J Mol Biol. 1976 Sep 25;106(3):857–869. doi: 10.1016/0022-2836(76)90269-2. [DOI] [PubMed] [Google Scholar]
  32. Platt T., Weber K., Ganem D., Miller J. H. Translational restarts: AUG reinitiation of a lac repressor fragment. Proc Natl Acad Sci U S A. 1972 Apr;69(4):897–901. doi: 10.1073/pnas.69.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Postle K., Nguyen T. T., Bertrand K. P. Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant. Nucleic Acids Res. 1984 Jun 25;12(12):4849–4863. doi: 10.1093/nar/12.12.4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sauer R. T., Krovatin W., DeAnda J., Youderian P., Susskind M. M. Primary structure of the immI immunity region of bacteriophage P22. J Mol Biol. 1983 Aug 25;168(4):699–713. doi: 10.1016/s0022-2836(83)80070-9. [DOI] [PubMed] [Google Scholar]
  36. Sauer R. T., Yocum R. R., Doolittle R. F., Lewis M., Pabo C. O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature. 1982 Jul 29;298(5873):447–451. doi: 10.1038/298447a0. [DOI] [PubMed] [Google Scholar]
  37. Shaw D. J., Rice D. W., Guest J. R. Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli. J Mol Biol. 1983 May 15;166(2):241–247. doi: 10.1016/s0022-2836(83)80011-4. [DOI] [PubMed] [Google Scholar]
  38. Steitz T. A., Ohlendorf D. H., McKay D. B., Anderson W. F., Matthews B. W. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins. Proc Natl Acad Sci U S A. 1982 May;79(10):3097–3100. doi: 10.1073/pnas.79.10.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  40. Weber I. T., McKay D. B., Steitz T. A. Two helix DNA binding motif of CAP found in lac repressor and gal repressor. Nucleic Acids Res. 1982 Aug 25;10(16):5085–5102. doi: 10.1093/nar/10.16.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yanofsky C., Horn V. Rifampin resistance mutations that alter the efficiency of transcription termination at the tryptophan operon attenuator. J Bacteriol. 1981 Mar;145(3):1334–1341. doi: 10.1128/jb.145.3.1334-1341.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yanofsky C., Kelley R. L., Horn V. Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J Bacteriol. 1984 Jun;158(3):1018–1024. doi: 10.1128/jb.158.3.1018-1024.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yanofsky C. Tryptophan biosynthesis in Escherichia coli. Genetic determination of the proteins involved. JAMA. 1971 Nov 15;218(7):1026–1035. [PubMed] [Google Scholar]
  44. Youderian P., Vershon A., Bouvier S., Sauer R. T., Susskind M. M. Changing the DNA-binding specificity of a repressor. Cell. 1983 Dec;35(3 Pt 2):777–783. doi: 10.1016/0092-8674(83)90110-1. [DOI] [PubMed] [Google Scholar]
  45. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES