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. 1995 May;177(9):2343–2353. doi: 10.1128/jb.177.9.2343-2353.1995

Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium.

M M Hryniewicz 1, N M Kredich 1
PMCID: PMC176890  PMID: 7730263

Abstract

CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

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Selected References

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  1. Bender R. A. The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes. Mol Microbiol. 1991 Nov;5(11):2575–2580. doi: 10.1111/j.1365-2958.1991.tb01965.x. [DOI] [PubMed] [Google Scholar]
  2. Bielińska A., Hulanicka D. Regulation of the cysB gene expression in Escherichia coli. Acta Biochim Pol. 1986;33(2):133–137. [PubMed] [Google Scholar]
  3. 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]
  4. Carra J. H., Schleif R. F. Variation of half-site organization and DNA looping by AraC protein. EMBO J. 1993 Jan;12(1):35–44. doi: 10.1002/j.1460-2075.1993.tb05629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang M., Crawford I. P. In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites. J Bacteriol. 1991 Mar;173(5):1590–1597. doi: 10.1128/jb.173.5.1590-1597.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang M., Crawford I. P. The roles of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res. 1990 Feb 25;18(4):979–988. doi: 10.1093/nar/18.4.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cho K., Winans S. C. Altered-function mutations in the Agrobacterium tumefaciens OccR protein and in an OccR-regulated promoter. J Bacteriol. 1993 Dec;175(23):7715–7719. doi: 10.1128/jb.175.23.7715-7719.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ebright R. H. Transcription activation at Class I CAP-dependent promoters. Mol Microbiol. 1993 May;8(5):797–802. doi: 10.1111/j.1365-2958.1993.tb01626.x. [DOI] [PubMed] [Google Scholar]
  9. Fisher R. F., Long S. R. Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA. J Mol Biol. 1993 Oct 5;233(3):336–348. doi: 10.1006/jmbi.1993.1515. [DOI] [PubMed] [Google Scholar]
  10. Giffard P. M., Booth I. R. The rpoA341 allele of Escherichia coli specifically impairs the transcription of a group of positively-regulated operons. Mol Gen Genet. 1988 Sep;214(1):148–152. doi: 10.1007/BF00340193. [DOI] [PubMed] [Google Scholar]
  11. Giffard P. M., Rowland G. C., Kroll R. G., Stewart L. M., Bakker E. P., Booth I. R. Phenotypic properties of a unique rpoA mutation (phs) of Escherichia coli. J Bacteriol. 1985 Nov;164(2):904–910. doi: 10.1128/jb.164.2.904-910.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goethals K., Van Montagu M., Holsters M. Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1646–1650. doi: 10.1073/pnas.89.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gussin G. N., Olson C., Igarashi K., Ishihama A. Activation defects caused by mutations in Escherichia coli rpoA are promoter specific. J Bacteriol. 1992 Aug;174(15):5156–5160. doi: 10.1128/jb.174.15.5156-5160.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hryniewicz M. M., Kredich N. M. Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium. J Bacteriol. 1994 Jun;176(12):3673–3682. doi: 10.1128/jb.176.12.3673-3682.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hryniewicz M. M., Kredich N. M. The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate. J Bacteriol. 1991 Sep;173(18):5876–5886. doi: 10.1128/jb.173.18.5876-5886.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang J. Z., Schell M. A. In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation. J Biol Chem. 1991 Jun 15;266(17):10830–10838. [PubMed] [Google Scholar]
  19. Igarashi K., Ishihama A. Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell. 1991 Jun 14;65(6):1015–1022. doi: 10.1016/0092-8674(91)90553-b. [DOI] [PubMed] [Google Scholar]
  20. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ishihama A. Role of the RNA polymerase alpha subunit in transcription activation. Mol Microbiol. 1992 Nov;6(22):3283–3288. doi: 10.1111/j.1365-2958.1992.tb02196.x. [DOI] [PubMed] [Google Scholar]
  22. Jagura-Burdzy G., Hulanicka D. Use of gene fusions to study expression of cysB, the regulatory gene of the cysteine regulon. J Bacteriol. 1981 Sep;147(3):744–751. doi: 10.1128/jb.147.3.744-751.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kredich N. M. The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli. Mol Microbiol. 1992 Oct;6(19):2747–2753. doi: 10.1111/j.1365-2958.1992.tb01453.x. [DOI] [PubMed] [Google Scholar]
  24. Lindquist S., Lindberg F., Normark S. Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene. J Bacteriol. 1989 Jul;171(7):3746–3753. doi: 10.1128/jb.171.7.3746-3753.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lombardo M. J., Bagga D., Miller C. G. Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium. J Bacteriol. 1991 Dec;173(23):7511–7518. doi: 10.1128/jb.173.23.7511-7518.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Maxon M. E., Wigboldus J., Brot N., Weissbach H. Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7076–7079. doi: 10.1073/pnas.87.18.7076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller B. E., Kredich N. M. Purification of the cysB protein from Salmonella typhimurium. J Biol Chem. 1987 May 5;262(13):6006–6009. [PubMed] [Google Scholar]
  29. Monroe R. S., Ostrowski J., Hryniewicz M. M., Kredich N. M. In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium. J Bacteriol. 1990 Dec;172(12):6919–6929. doi: 10.1128/jb.172.12.6919-6929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ostrowski J., Kredich N. M. In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide. J Bacteriol. 1990 Feb;172(2):779–785. doi: 10.1128/jb.172.2.779-785.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ostrowski J., Kredich N. M. Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by cysB protein and N-acetyl-L-serine. J Bacteriol. 1989 Jan;171(1):130–140. doi: 10.1128/jb.171.1.130-140.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ostrowski J., Kredich N. M. Negative autoregulation of cysB in Salmonella typhimurium: in vitro interactions of CysB protein with the cysB promoter. J Bacteriol. 1991 Apr;173(7):2212–2218. doi: 10.1128/jb.173.7.2212-2218.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  34. Parsek M. R., Shinabarger D. L., Rothmel R. K., Chakrabarty A. M. Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. J Bacteriol. 1992 Dec;174(23):7798–7806. doi: 10.1128/jb.174.23.7798-7806.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schell M. A., Brown P. H., Raju S. Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator. J Biol Chem. 1990 Mar 5;265(7):3844–3850. [PubMed] [Google Scholar]
  36. 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]
  37. Shi X., Bennett G. N. Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli. J Bacteriol. 1994 Nov;176(22):7017–7023. doi: 10.1128/jb.176.22.7017-7023.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sirko A., Hryniewicz M., Hulanicka D., Böck A. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. J Bacteriol. 1990 Jun;172(6):3351–3357. doi: 10.1128/jb.172.6.3351-3357.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tao K., Fujita N., Ishihama A. Involvement of the RNA polymerase alpha subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein. Mol Microbiol. 1993 Mar;7(6):859–864. doi: 10.1111/j.1365-2958.1993.tb01176.x. [DOI] [PubMed] [Google Scholar]
  40. Tartaglia L. A., Gimeno C. J., Storz G., Ames B. N. Multidegenerate DNA recognition by the OxyR transcriptional regulator. J Biol Chem. 1992 Jan 25;267(3):2038–2045. [PubMed] [Google Scholar]
  41. Thomas M. S., Glass R. E. Escherichia coli rpoA mutation which impairs transcription of positively regulated systems. Mol Microbiol. 1991 Nov;5(11):2719–2725. doi: 10.1111/j.1365-2958.1991.tb01980.x. [DOI] [PubMed] [Google Scholar]
  42. Thöny B., Hwang D. S., Fradkin L., Kornberg A. iciA, an Escherichia coli gene encoding a specific inhibitor of chromosomal initiation of replication in vitro. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4066–4070. doi: 10.1073/pnas.88.10.4066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Urbanowski M. L., Stauffer G. V. Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium. J Bacteriol. 1989 Oct;171(10):5620–5629. doi: 10.1128/jb.171.10.5620-5629.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang S. P., Stacey G. Studies of the Bradyrhizobium japonicum nodD1 promoter: a repeated structure for the nod box. J Bacteriol. 1991 Jun;173(11):3356–3365. doi: 10.1128/jb.173.11.3356-3365.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Zhou Y., Zhang X., Ebright R. H. Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6081–6085. doi: 10.1073/pnas.90.13.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zou C., Fujita N., Igarashi K., Ishihama A. Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase. Mol Microbiol. 1992 Sep;6(18):2599–2605. doi: 10.1111/j.1365-2958.1992.tb01437.x. [DOI] [PubMed] [Google Scholar]

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