Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jul;177(14):4113–4120. doi: 10.1128/jb.177.14.4113-4120.1995

Characterization of the MetR binding sites for the glyA gene of Escherichia coli.

E Lorenz 1, G V Stauffer 1
PMCID: PMC177144  PMID: 7608086

Abstract

Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein. Gel shift assays and DNase I protection assays verified that both sites bind MetR. Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region. The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees. MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression. Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation.

Full Text

The Full Text of this article is available as a PDF (828.3 KB).

Selected References

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

  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. Byerly K. A., Urbanowski M. L., Stauffer G. V. The metR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation. J Bacteriol. 1991 Jun;173(11):3547–3553. doi: 10.1128/jb.173.11.3547-3553.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Costa L. E., Reynafarje B., Lehninger A. L. Stoichiometry of mitochondrial H+ translocation coupled to succinate oxidation at level flow. J Biol Chem. 1984 Apr 25;259(8):4802–4811. [PubMed] [Google Scholar]
  5. Cowan J. M., Urbanowski M. L., Talmi M., Stauffer G. V. Regulation of the Salmonella typhimurium metF gene by the MetR protein. J Bacteriol. 1993 Sep;175(18):5862–5866. doi: 10.1128/jb.175.18.5862-5866.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greene R. C., Radovich C. Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli. J Bacteriol. 1975 Oct;124(1):269–278. doi: 10.1128/jb.124.1.269-278.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hagerman P. J. Sequence-directed curvature of DNA. Annu Rev Biochem. 1990;59:755–781. doi: 10.1146/annurev.bi.59.070190.003543. [DOI] [PubMed] [Google Scholar]
  10. Kilstrup M., Meng L. M., Neuhard J., Nygaard P. Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli. J Bacteriol. 1989 Apr;171(4):2124–2127. doi: 10.1128/jb.171.4.2124-2127.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Mansouri A., Decter J. B., Silber R. Studies on the regulation of one-carbon metabolism. II. Repression-derepression of serine hydroxymethyltransferase by methionine in Escherichia coli 113-3. J Biol Chem. 1972 Jan 25;247(2):348–352. [PubMed] [Google Scholar]
  14. Mares R., Urbanowski M. L., Stauffer G. V. Regulation of the Salmonella typhimurium metA gene by the metR protein and homocysteine. J Bacteriol. 1992 Jan;174(2):390–397. doi: 10.1128/jb.174.2.390-397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  16. Meedel T. H., Pizer L. I. Regulation of one-carbon biosynthesis and utilization in Escherichia coli. J Bacteriol. 1974 Jun;118(3):905–910. doi: 10.1128/jb.118.3.905-910.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Plamann M. D., Stauffer G. V. Characterization of the Escherichia coli gene for serine hydroxymethyltransferase. Gene. 1983 Apr;22(1):9–18. doi: 10.1016/0378-1119(83)90059-8. [DOI] [PubMed] [Google Scholar]
  18. Plamann M. D., Stauffer G. V. Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine. J Bacteriol. 1989 Sep;171(9):4958–4962. doi: 10.1128/jb.171.9.4958-4962.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Plamann M. D., Stauffer L. T., Urbanowski M. L., Stauffer G. V. Complete nucleotide sequence of the E. coli glyA gene. Nucleic Acids Res. 1983 Apr 11;11(7):2065–2075. doi: 10.1093/nar/11.7.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rolfes R. J., Zalkin H. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator. J Biol Chem. 1988 Dec 25;263(36):19653–19661. [PubMed] [Google Scholar]
  21. 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]
  22. Schell M. A. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol. 1993;47:597–626. doi: 10.1146/annurev.mi.47.100193.003121. [DOI] [PubMed] [Google Scholar]
  23. Schmitz A., Galas D. J. The interaction of RNA polymerase and lac repressor with the lac control region. Nucleic Acids Res. 1979 Jan;6(1):111–137. doi: 10.1093/nar/6.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shimada K., Weisberg R. A., Gottesman M. E. Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J Mol Biol. 1972 Feb 14;63(3):483–503. doi: 10.1016/0022-2836(72)90443-3. [DOI] [PubMed] [Google Scholar]
  25. Stauffer G. V., Brenchley J. E. Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2. J Bacteriol. 1977 Feb;129(2):740–749. doi: 10.1128/jb.129.2.740-749.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Steiert J. G., Kubu C., Stauffer G. V. The PurR binding site in the glyA promoter region of Escherichia coli. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):299–304. doi: 10.1016/0378-1097(92)90044-o. [DOI] [PubMed] [Google Scholar]
  27. Steiert J. G., Rolfes R. J., Zalkin H., Stauffer G. V. Regulation of the Escherichia coli glyA gene by the purR gene product. J Bacteriol. 1990 Jul;172(7):3799–3803. doi: 10.1128/jb.172.7.3799-3803.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Steiert J. G., Urbanowski M. L., Stauffer L. T., Plamann M. D., Stauffer G. V. Nucleotide sequence of the Salmonella typhimurium glyA gene. DNA Seq. 1990;1(2):107–113. doi: 10.3109/10425179009016038. [DOI] [PubMed] [Google Scholar]
  29. Taylor R. T., Dickerman H., Weissbach H. Control of one-carbon metabolism in a methionine-B12 auxotroph of Escherichia coli. Arch Biochem Biophys. 1966 Nov;117(2):405–412. doi: 10.1016/0003-9861(66)90429-2. [DOI] [PubMed] [Google Scholar]
  30. Urbanowski M. L., Stauffer G. V. Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene. J Bacteriol. 1986 Mar;165(3):740–745. doi: 10.1128/jb.165.3.740-745.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Urbanowski M. L., Stauffer G. V. Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol. 1989 Jun;171(6):3277–3281. doi: 10.1128/jb.171.6.3277-3281.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Urbanowski M. L., Stauffer L. T., Plamann L. S., Stauffer G. V. A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J Bacteriol. 1987 Apr;169(4):1391–1397. doi: 10.1128/jb.169.4.1391-1397.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  34. van der Vliet P. C., Verrijzer C. P. Bending of DNA by transcription factors. Bioessays. 1993 Jan;15(1):25–32. doi: 10.1002/bies.950150105. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES