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. 1985 Jul;163(1):376–384. doi: 10.1128/jb.163.1.376-384.1985

Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12.

K F Wertman, D W Mount
PMCID: PMC219124  PMID: 3891738

Abstract

The specificity of LexA protein binding was investigated by quantifying the repressibility of several mutant recA and lexA operator-promoter regions fused to the Escherichia coli galactokinase (galK) gene. The results of this analysis indicate that two sets of four nucleotides, one set at each end of the operator (terminal-nucleotide contacts), are most critical for repressor binding. In addition, our results suggest that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator have comparable effects on repressibility. The symmetry of this interaction justified reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus (5')CTGTATAT. Although the first four positions of this sequence were most important, the last four were well conserved among binding sites and appeared to modulate repressor affinity. The role of the terminal-nucleotide contacts and the mechanism by which the internal sequences affected repressor binding are discussed.

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Selected References

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

  1. 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]
  2. Brent R., Ptashne M. Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4204–4208. doi: 10.1073/pnas.78.7.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Champoux J. J. Proteins that affect DNA conformation. Annu Rev Biochem. 1978;47:449–479. doi: 10.1146/annurev.bi.47.070178.002313. [DOI] [PubMed] [Google Scholar]
  4. Clark A. J. recA operator mutations and their usefulness. Biochimie. 1982 Aug-Sep;64(8-9):669–675. doi: 10.1016/s0300-9084(82)80108-9. [DOI] [PubMed] [Google Scholar]
  5. Dickerson R. E., Drew H. R. Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure. J Mol Biol. 1981 Jul 15;149(4):761–786. doi: 10.1016/0022-2836(81)90357-0. [DOI] [PubMed] [Google Scholar]
  6. Drew H. R., Wing R. M., Takano T., Broka C., Tanaka S., Itakura K., Dickerson R. E. Structure of a B-DNA dodecamer: conformation and dynamics. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2179–2183. doi: 10.1073/pnas.78.4.2179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ginsburg H., Edmiston S. H., Harper J., Mount D. W. Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12. Mol Gen Genet. 1982;187(1):4–11. doi: 10.1007/BF00384376. [DOI] [PubMed] [Google Scholar]
  8. Hogan M., LeGrange J., Austin B. Dependence of DNA helix flexibility on base composition. Nature. 1983 Aug 25;304(5928):752–754. doi: 10.1038/304752a0. [DOI] [PubMed] [Google Scholar]
  9. Huisman O., D'Ari R., Casaregola S. How Escherichia coli sets different basal levels in SOS operons. Biochimie. 1982 Aug-Sep;64(8-9):709–712. doi: 10.1016/s0300-9084(82)80115-6. [DOI] [PubMed] [Google Scholar]
  10. Hübscher U., Lutz H., Kornberg A. Novel histone H2A-like protein of escherichia coli. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5097–5101. doi: 10.1073/pnas.77.9.5097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  12. Little J. W. Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1375–1379. doi: 10.1073/pnas.81.5.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  14. Little J. W., Mount D. W., Yanisch-Perron C. R. Purified lexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4199–4203. doi: 10.1073/pnas.78.7.4199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lomonossoff G. P., Butler P. J., Klug A. Sequence-dependent variation in the conformation of DNA. J Mol Biol. 1981 Jul 15;149(4):745–760. doi: 10.1016/0022-2836(81)90356-9. [DOI] [PubMed] [Google Scholar]
  16. Mankovich J. A., Lai P. H., Gokul N., Konisky J. Organization of the colicin Ib gene. Promoter structure and immunity domain. J Biol Chem. 1984 Jul 25;259(14):8764–8768. [PubMed] [Google Scholar]
  17. Markham B. E., Harper J. E., Mount D. W. Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction. Mol Gen Genet. 1985;198(2):207–212. doi: 10.1007/BF00382997. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. McKenney K., Shimatake H., Court D., Schmeissner U., Brady C., Rosenberg M. A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. Gene Amplif Anal. 1981;2:383–415. [PubMed] [Google Scholar]
  20. McPartland A., Green L., Echols H. Control of recA gene RNA in E. coli: regulatory and signal genes. Cell. 1980 Jul;20(3):731–737. doi: 10.1016/0092-8674(80)90319-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mizusawa S., Gottesman S. Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci U S A. 1983 Jan;80(2):358–362. doi: 10.1073/pnas.80.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mount D. W. A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci U S A. 1977 Jan;74(1):300–304. doi: 10.1073/pnas.74.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pabo C. O., Krovatin W., Jeffrey A., Sauer R. T. The N-terminal arms of lambda repressor wrap around the operator DNA. Nature. 1982 Jul 29;298(5873):441–443. doi: 10.1038/298441a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Record M. T., Jr, Lohman M. L., De Haseth P. Ion effects on ligand-nucleic acid interactions. J Mol Biol. 1976 Oct 25;107(2):145–158. doi: 10.1016/s0022-2836(76)80023-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shortle D., Nathans D. Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc Natl Acad Sci U S A. 1978 May;75(5):2170–2174. doi: 10.1073/pnas.75.5.2170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wertman K. F., Little J. W., Mount D. W. Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3801–3805. doi: 10.1073/pnas.81.12.3801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van den Elzen P. J., Maat J., Walters H. H., Veltkamp E., Nijkamp H. J. The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity. Nucleic Acids Res. 1982 Mar 25;10(6):1913–1928. doi: 10.1093/nar/10.6.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]

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