Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 May;174(9):2763–2770. doi: 10.1128/jb.174.9.2763-2770.1992

Promoter elements required for positive control of transcription of the Escherichia coli uhpT gene.

T J Merkel 1, D M Nelson 1, C L Brauer 1, R J Kadner 1
PMCID: PMC205926  PMID: 1569008

Abstract

The uhpABCT locus of Escherichia coli encodes the transport system which allows the cell to accumulate a variety of sugar phosphates in unaltered form. The expression of uhpT, the gene encoding the transport protein, is regulated by the uhpABC gene products. The UhpA protein is required for expression; its deduced amino acid sequence shows that it belongs to a subfamily of bacterial transcription regulators including NarL, DegU, and FixJ. Members of this subfamily have an amino-terminal phosphorylation domain characteristic of so-called two-component regulators, such as OmpR, CheY, PhoB, and NtrC, and a carboxyl-terminal domain conserved among many transcriptional activators, including LuxR and MalT. The major sequence elements in the uhpT promoter that are needed for uhpT expression were investigated. Northern (RNA) hybridization analysis showed that the uhpT transcript was only present in cells induced for UhpT transport activity. The start site of transcription was identified by primer extension. Comparison of the regions upstream of the uhpT transcription start site in E. coli and Salmonella typhimurium suggested the presence of four sequence elements that might be involved in promoter function: a typical -10 region, a short inverted repeat centered at -32, a long inverted repeat centered at -64, and a cyclic AMP receptor protein-binding sequence centered at -103. Deletion and linker substitution mutations in the promoter demonstrated that the presence of the cyclic AMP receptor protein-binding site resulted in about an eightfold increase in promoter activity and that the -64, -32, and -10 elements were essential for promoter function. In vivo titration of transcriptional activator UhpA by the intact or mutant promoters on multicopy plasmids identified the -64 element as the UhpA-binding site. The two halves of the -64 inverted repeat did not contribute equally to promoter function and did not have to be intact for UhpA titration. The sequence recognized by UhpA is predicted to be 5' -GGCAAAACNNNGAAA.

Full text

PDF
2763

Images in this article

2766Image
on p.2766

Selected References

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

  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Alper M. D., Ames B. N. Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants. J Bacteriol. 1978 Jan;133(1):149–157. doi: 10.1128/jb.133.1.149-157.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dietz G. W., Jr The hexose phosphate transport system of Escherichia coli. Adv Enzymol Relat Areas Mol Biol. 1976;44:237–259. doi: 10.1002/9780470122891.ch7. [DOI] [PubMed] [Google Scholar]
  6. Dunlap P. V., Greenberg E. P. Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit. J Bacteriol. 1988 Sep;170(9):4040–4046. doi: 10.1128/jb.170.9.4040-4046.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferenci T., Kornberg H. L., Smith Janet. Isolation and properties of a regulatory mutant in the hexose phosphate transport system of Escherichia coli. FEBS Lett. 1971 Mar 5;13(3):133–136. doi: 10.1016/0014-5793(71)80218-1. [DOI] [PubMed] [Google Scholar]
  8. Gaston K., Bell A., Kolb A., Buc H., Busby S. Stringent spacing requirements for transcription activation by CRP. Cell. 1990 Aug 24;62(4):733–743. doi: 10.1016/0092-8674(90)90118-x. [DOI] [PubMed] [Google Scholar]
  9. Henikoff S., Wallace J. C., Brown J. P. Finding protein similarities with nucleotide sequence databases. Methods Enzymol. 1990;183:111–132. doi: 10.1016/0076-6879(90)83009-x. [DOI] [PubMed] [Google Scholar]
  10. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  11. Island M. D., Wei B. Y., Kadner R. J. Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium. J Bacteriol. 1992 May;174(9):2754–2762. doi: 10.1128/jb.174.9.2754-2762.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Li S. F., DeMoss J. A. Location of sequences in the nar promoter of Escherichia coli required for regulation by Fnr and NarL. J Biol Chem. 1988 Sep 25;263(27):13700–13705. [PubMed] [Google Scholar]
  13. Mandecki W., Caruthers M. H. Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region. Gene. 1984 Nov;31(1-3):263–267. doi: 10.1016/0378-1119(84)90219-1. [DOI] [PubMed] [Google Scholar]
  14. Msadek T., Kunst F., Klier A., Rapoport G. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J Bacteriol. 1991 Apr;173(7):2366–2377. doi: 10.1128/jb.173.7.2366-2377.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pogell B. M., Maity B. R., Frumkin S., Shapiro S. Induction of an active transport system for glucose 6-phosphate in Escherichia coli. Arch Biochem Biophys. 1966 Sep 26;116(1):406–415. doi: 10.1016/0003-9861(66)90047-6. [DOI] [PubMed] [Google Scholar]
  16. Raibaud O., Vidal-Ingigliardi D., Richet E. A complex nucleoprotein structure involved in activation of transcription of two divergent Escherichia coli promoters. J Mol Biol. 1989 Feb 5;205(3):471–485. doi: 10.1016/0022-2836(89)90218-0. [DOI] [PubMed] [Google Scholar]
  17. Richet E., Vidal-Ingigliardi D., Raibaud O. A new mechanism for coactivation of transcription initiation: repositioning of an activator triggered by the binding of a second activator. Cell. 1991 Sep 20;66(6):1185–1195. doi: 10.1016/0092-8674(91)90041-v. [DOI] [PubMed] [Google Scholar]
  18. Roland K. L., Liu C. G., Turnbough C. L., Jr Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7149–7153. doi: 10.1073/pnas.85.19.7149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  20. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  21. Shadel G. S., Baldwin T. O. The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene. J Bacteriol. 1991 Jan;173(2):568–574. doi: 10.1128/jb.173.2.568-574.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shattuck-Eidens D. M., Kadner R. J. Exogenous induction of the Escherichia coli hexose phosphate transport system defined by uhp-lac operon fusions. J Bacteriol. 1981 Oct;148(1):203–209. doi: 10.1128/jb.148.1.203-209.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shattuck-Eidens D. M., Kadner R. J. Molecular cloning of the uhp region and evidence for a positive activator for expression of the hexose phosphate transport system of Escherichia coli. J Bacteriol. 1983 Sep;155(3):1062–1070. doi: 10.1128/jb.155.3.1062-1070.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  25. Stock A. M., Mottonen J. M., Stock J. B., Schutt C. E. Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature. 1989 Feb 23;337(6209):745–749. doi: 10.1038/337745a0. [DOI] [PubMed] [Google Scholar]
  26. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stout V., Torres-Cabassa A., Maurizi M. R., Gutnick D., Gottesman S. RcsA, an unstable positive regulator of capsular polysaccharide synthesis. J Bacteriol. 1991 Mar;173(5):1738–1747. doi: 10.1128/jb.173.5.1738-1747.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ushida C., Aiba H. Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. Nucleic Acids Res. 1990 Nov 11;18(21):6325–6330. doi: 10.1093/nar/18.21.6325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vidal-Ingigliardi D., Raibaud O. Three adjacent binding sites for cAMP receptor protein are involved in the activation of the divergent malEp-malKp promoters. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):229–233. doi: 10.1073/pnas.88.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vidal-Ingigliardi D., Richet E., Raibaud O. Two MalT binding sites in direct repeat. A structural motif involved in the activation of all the promoters of the maltose regulons in Escherichia coli and Klebsiella pneumoniae. J Mol Biol. 1991 Mar 20;218(2):323–334. doi: 10.1016/0022-2836(91)90715-i. [DOI] [PubMed] [Google Scholar]
  31. Weston L. A., Kadner R. J. Identification of uhp polypeptides and evidence for their role in exogenous induction of the sugar phosphate transport system of Escherichia coli K-12. J Bacteriol. 1987 Aug;169(8):3546–3555. doi: 10.1128/jb.169.8.3546-3555.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weston L. A., Kadner R. J. Role of uhp genes in expression of the Escherichia coli sugar-phosphate transport system. J Bacteriol. 1988 Aug;170(8):3375–3383. doi: 10.1128/jb.170.8.3375-3383.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Winkler H. H. Compartmentation in the induction of the hexose-6-phosphate transport system of Escherichia coli. J Bacteriol. 1970 Feb;101(2):470–475. doi: 10.1128/jb.101.2.470-475.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  35. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]

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

RESOURCES