Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Oct;171(10):5620–5629. doi: 10.1128/jb.171.10.5620-5629.1989

Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium.

M L Urbanowski 1, G V Stauffer 1
PMCID: PMC210406  PMID: 2676984

Abstract

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.

Full text

PDF
5626

Images in this article

Selected References

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

  1. Ahmed A. Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli. Gene. 1984 Apr;28(1):37–43. doi: 10.1016/0378-1119(84)90085-4. [DOI] [PubMed] [Google Scholar]
  2. Baker R. F., Yanofsky C. The periodicity of RNA polymerase initiations: a new regulatory feature of transcription. Proc Natl Acad Sci U S A. 1968 May;60(1):313–320. doi: 10.1073/pnas.60.1.313. [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. Dev I. K., Harvey R. J. Regulation of synthesis of serine hydroxymethyltransferase in chemostat cultures of Escherichia coli. J Biol Chem. 1984 Jul 10;259(13):8394–8401. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Graña D., Gardella T., Susskind M. M. The effects of mutations in the ant promoter of phage P22 depend on context. Genetics. 1988 Oct;120(2):319–327. doi: 10.1093/genetics/120.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Martin K., Huo L., Schleif R. F. The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3654–3658. doi: 10.1073/pnas.83.11.3654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  14. Plamann L. S., Stauffer G. V. Nucleotide sequence of the Salmonella typhimurium metR gene and the metR-metE control region. J Bacteriol. 1987 Sep;169(9):3932–3937. doi: 10.1128/jb.169.9.3932-3937.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Plamann L. S., Urbanowski M. L., Stauffer G. V. Salmonella typhimurium metE operator-constitutive mutations. Gene. 1988 Dec 15;73(1):201–208. doi: 10.1016/0378-1119(88)90326-5. [DOI] [PubMed] [Google Scholar]
  16. Rymond B. C., Zitomer R. S., Schümperli D., Rosenberg M. The expression in yeast of the Escherichia coli galK gene on CYC1::galK fusion plasmids. Gene. 1983 Nov;25(2-3):249–262. doi: 10.1016/0378-1119(83)90229-9. [DOI] [PubMed] [Google Scholar]
  17. Saint-Girons I., Parsot C., Zakin M. M., Bârzu O., Cohen G. N. Methionine biosynthesis in Enterobacteriaceae: biochemical, regulatory, and evolutionary aspects. CRC Crit Rev Biochem. 1988;23 (Suppl 1):S1–42. doi: 10.3109/10409238809083374. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Stauffer G. V., Plamann M. D., Stauffer L. T. Construction and expression of hybrid plasmids containing the Escherichia coli glyA genes. Gene. 1981 Jun-Jul;14(1-2):63–72. doi: 10.1016/0378-1119(81)90148-7. [DOI] [PubMed] [Google Scholar]
  21. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Urbanowski M. L., Stauffer G. V. Regulation of the metR gene of Salmonella typhimurium. J Bacteriol. 1987 Dec;169(12):5841–5844. doi: 10.1128/jb.169.12.5841-5844.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Urbanowski M. L., Stauffer G. V. The control region of the metH gene of Salmonella typhimurium LT2: an atypical met promoter. Gene. 1988 Dec 15;73(1):193–200. doi: 10.1016/0378-1119(88)90325-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Wek R. C., Hatfield G. W. Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. J Mol Biol. 1988 Oct 5;203(3):643–663. doi: 10.1016/0022-2836(88)90199-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES