Skip to main content
PMC home page
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
. 1991 Aug 15;88(16):7356–7360. doi: 10.1073/pnas.88.16.7356

Mutant LexA proteins with an increased rate of in vivo cleavage.

M H Smith 1, M M Cavenagh 1, J W Little 1
PMCID: PMC52294  PMID: 1908093

Abstract

LexA repressor of Escherichia coli is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo. This cleavage reaction can proceed in vitro in the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH. Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in vivo, compared with wild-type LexA. Efficient in vivo cleavage of these mutant proteins also took place without RecA. Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion. These mutations changed Gln-92, which lies near the cleavage site, to tyrosine, phenylalanine, or tryptophan. The latter mutation increased the rate of cleavage approximately 500-fold. These findings imply that the rate of wild-type LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNA-damaging treatments. Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.

Full text

PDF
7356

Images in this article

7358Image
on p.7358
7359Image
on p.7359
7359Image
on p.7359
7359Image
on p.7359

Selected References

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

  1. Booth I. R. Regulation of cytoplasmic pH in bacteria. Microbiol Rev. 1985 Dec;49(4):359–378. doi: 10.1128/mr.49.4.359-378.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burckhardt S. E., Woodgate R., Scheuermann R. H., Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burley S. K., Petsko G. A. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science. 1985 Jul 5;229(4708):23–28. doi: 10.1126/science.3892686. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  5. Craig N. L., Roberts J. W. E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide. Nature. 1980 Jan 3;283(5742):26–30. doi: 10.1038/283026a0. [DOI] [PubMed] [Google Scholar]
  6. Csonka L. N., Clark A. J. Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome. Genetics. 1979 Oct;93(2):321–343. doi: 10.1093/genetics/93.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gimble F. S., Sauer R. T. Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions. J Mol Biol. 1986 Nov 5;192(1):39–47. doi: 10.1016/0022-2836(86)90462-6. [DOI] [PubMed] [Google Scholar]
  8. Hahn C. S., Strauss J. H. Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease. J Virol. 1990 Jun;64(6):3069–3073. doi: 10.1128/jvi.64.6.3069-3073.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hunter T. A tail of two src's: mutatis mutandis. Cell. 1987 Apr 10;49(1):1–4. doi: 10.1016/0092-8674(87)90745-8. [DOI] [PubMed] [Google Scholar]
  10. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. LeGendre N., Matsudaira P. Direct protein microsequencing from Immobilon-P Transfer Membrane. Biotechniques. 1988 Feb;6(2):154–159. [PubMed] [Google Scholar]
  12. Lin L. L., Little J. W. Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins. J Mol Biol. 1989 Dec 5;210(3):439–452. doi: 10.1016/0022-2836(89)90121-6. [DOI] [PubMed] [Google Scholar]
  13. Lin L. L., Little J. W. Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2163–2173. doi: 10.1128/jb.170.5.2163-2173.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Little J. W., Harper J. E. Identification of the lexA gene product of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6147–6151. doi: 10.1073/pnas.76.12.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Little J. W. Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12. Gene. 1980 Aug;10(3):237–247. doi: 10.1016/0378-1119(80)90053-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Little J. W. The SOS regulatory system: control of its state by the level of RecA protease. J Mol Biol. 1983 Jul 15;167(4):791–808. doi: 10.1016/s0022-2836(83)80111-9. [DOI] [PubMed] [Google Scholar]
  19. Ossanna N., Mount D. W. Mutations in uvrD induce the SOS response in Escherichia coli. J Bacteriol. 1989 Jan;171(1):303–307. doi: 10.1128/jb.171.1.303-307.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pacelli L. Z., Edmiston S. H., Mount D. W. Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. J Bacteriol. 1979 Jan;137(1):568–573. doi: 10.1128/jb.137.1.568-573.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roland K. L., Little J. W. Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model. J Biol Chem. 1990 Aug 5;265(22):12828–12835. [PubMed] [Google Scholar]
  22. Slilaty S. N., Little J. W. Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3987–3991. doi: 10.1073/pnas.84.12.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Slilaty S. N., Rupley J. A., Little J. W. Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent. Biochemistry. 1986 Nov 4;25(22):6866–6875. doi: 10.1021/bi00370a020. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]
  26. Zilberstein D., Agmon V., Schuldiner S., Padan E. Escherichia coli intracellular pH, membrane potential, and cell growth. J Bacteriol. 1984 Apr;158(1):246–252. doi: 10.1128/jb.158.1.246-252.1984. [DOI] [PMC free article] [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