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. 1993 Feb;175(4):1110–1117. doi: 10.1128/jb.175.4.1110-1117.1993

Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine.

J V Platko 1, J M Calvo 1
PMCID: PMC193027  PMID: 8432705

Abstract

Lrp is a regulatory protein in Escherichia coli that increases expression of some operons and decreases expression of others. Mutations in Lrp were isolated on the basis of their effects on ilvIH, one of the operons regulated positively by Lrp. The ilvIH operon encodes an enzyme involved in the biosynthesis of leucine, valine, and isoleucine, and expression of this operon is repressed when cells are grown in the presence of leucine. Three groups of mutants were isolated. Mutant strains that were resistant to the repressive effects of leucine were termed leucine response mutants. These mutants had changes in the Lrp amino acid sequence between amino acid residues 108 and 149. Mutant strains having low expression of ilvIH in vivo were identified as colonies having reduced expression of a reporter gene. For some of these mutants, called DNA-binding mutants, binding to ilvIH DNA in vitro was markedly reduced. The mutations in these strains caused changes in Lrp between amino acids 16 and 70. Six of ten of these mutations were within a region having a putative helix-turn-helix motif. A third group of mutants had low ilvIH expression in vivo but apparently normal DNA binding in vitro. These mutants were called activation mutants since they affected the ability of Lrp to activate expression. Lrp from these strains had changes in amino acids between residues 76 and 125. This study suggests that Lrp has separate domains responsible for binding DNA, activating transcription, and responding to leucine.

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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 J. J., Quay S. C., Oxender D. L. Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12. J Bacteriol. 1976 Apr;126(1):80–90. doi: 10.1128/jb.126.1.80-90.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  3. Dodd I. B., Egan J. B. Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins. J Mol Biol. 1987 Apr 5;194(3):557–564. doi: 10.1016/0022-2836(87)90681-4. [DOI] [PubMed] [Google Scholar]
  4. Ernsting B. R., Atkinson M. R., Ninfa A. J., Matthews R. G. Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli. J Bacteriol. 1992 Feb;174(4):1109–1118. doi: 10.1128/jb.174.4.1109-1118.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  6. Haney S. A., Platko J. V., Oxender D. L., Calvo J. M. Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli. J Bacteriol. 1992 Jan;174(1):108–115. doi: 10.1128/jb.174.1.108-115.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haughn G. W., Squires C. H., De Felice M., Largo C. T., Calvo J. M. Unusual organization of the ilvIH promoter of Escherichia coli. J Bacteriol. 1985 Jul;163(1):186–198. doi: 10.1128/jb.163.1.186-198.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kölling R., Lother H. AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli. J Bacteriol. 1985 Oct;164(1):310–315. doi: 10.1128/jb.164.1.310-315.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lin R., D'Ari R., Newman E. B. Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine. J Bacteriol. 1992 Mar;174(6):1948–1955. doi: 10.1128/jb.174.6.1948-1955.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mallick U., Herrlich P. Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5520–5523. doi: 10.1073/pnas.76.11.5520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Newman E. B., D'Ari R., Lin R. T. The leucine-Lrp regulon in E. coli: a global response in search of a raison d'être. Cell. 1992 Feb 21;68(4):617–619. doi: 10.1016/0092-8674(92)90135-y. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Platko J. V., Willins D. A., Calvo J. M. The ilvIH operon of Escherichia coli is positively regulated. J Bacteriol. 1990 Aug;172(8):4563–4570. doi: 10.1128/jb.172.8.4563-4570.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  15. Ricca E., Aker D. A., Calvo J. M. A protein that binds to the regulatory region of the Escherichia coli ilvIH operon. J Bacteriol. 1989 Mar;171(3):1658–1664. doi: 10.1128/jb.171.3.1658-1664.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenthal E. R., Calvo J. M. Transcription termination sites at the distal end of the leu operon of Salmonella typhimurium. J Mol Biol. 1987 Apr 5;194(3):443–452. doi: 10.1016/0022-2836(87)90673-5. [DOI] [PubMed] [Google Scholar]
  17. Shestopalov B. V. Amino acid sequence template useful for alpha-helix-turn-alpha-helix prediction. FEBS Lett. 1988 Jun 6;233(1):105–108. doi: 10.1016/0014-5793(88)81364-4. [DOI] [PubMed] [Google Scholar]
  18. Squires C. H., De Felice M., Lago C. T., Calvo J. M. IlvHI locus of Salmonella typhimurium. J Bacteriol. 1983 Jun;154(3):1054–1063. doi: 10.1128/jb.154.3.1054-1063.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Squires C. H., De Felice M., Wessler S. R., Calvo J. M. Physical characterization of the ilvHI operon of Escherichia coli K-12. J Bacteriol. 1981 Sep;147(3):797–804. doi: 10.1128/jb.147.3.797-804.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tuan L. R., D'Ari R., Newman E. B. The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons. J Bacteriol. 1990 Aug;172(8):4529–4535. doi: 10.1128/jb.172.8.4529-4535.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ursini M. V., Arcari P., De Felice M. Acetohydroxy acid synthase isoenzymes of Escherichia coli K-12: a trans-acting regulatory locus of ilvHI gene expression. Mol Gen Genet. 1981;181(4):491–496. doi: 10.1007/BF00428741. [DOI] [PubMed] [Google Scholar]
  22. Willins D. A., Ryan C. W., Platko J. V., Calvo J. M. Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine. J Biol Chem. 1991 Jun 15;266(17):10768–10774. [PubMed] [Google Scholar]
  23. de Felice M., Lago C. T., Squires C. H., Calvo J. M. Acetohydroxy acid synthase isoenzymes of Escherichia coli K12 and Salmonella typhimurium. Ann Microbiol (Paris) 1982 Mar-Apr;133(2):251–256. [PubMed] [Google Scholar]
  24. de Wind N., de Jong M., Meijer M., Stuitje A. R. Site-directed mutagenesis of the Escherichia coli chromosome near oriC: identification and characterization of asnC, a regulatory element in E. coli asparagine metabolism. Nucleic Acids Res. 1985 Dec 20;13(24):8797–8811. doi: 10.1093/nar/13.24.8797. [DOI] [PMC free article] [PubMed] [Google Scholar]

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