Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Sep;176(17):5513–5524. doi: 10.1128/jb.176.17.5513-5524.1994

Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH.

R Osuna 1, B K Janes 1, R A Bender 1
PMCID: PMC196740  PMID: 8071230

Abstract

The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the catabolite gene activator protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.

Full text

PDF
5523

Images in this article

5516Image
on p.5516
5518Image
on p.5518
5519Image
on p.5519
5520Image
on p.5520

Selected References

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

  1. Aiba H. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 1983 Jan;32(1):141–149. doi: 10.1016/0092-8674(83)90504-4. [DOI] [PubMed] [Google Scholar]
  2. Aiba H., Nakamura T., Mitani H., Mori H. Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3329–3332. doi: 10.1002/j.1460-2075.1985.tb04084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball C. A., Osuna R., Ferguson K. C., Johnson R. C. Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. J Bacteriol. 1992 Dec;174(24):8043–8056. doi: 10.1128/jb.174.24.8043-8056.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell A., Gaston K., Williams R., Chapman K., Kolb A., Buc H., Minchin S., Williams J., Busby S. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 1990 Dec 25;18(24):7243–7250. doi: 10.1093/nar/18.24.7243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Botsford J. L., Harman J. G. Cyclic AMP in prokaryotes. Microbiol Rev. 1992 Mar;56(1):100–122. doi: 10.1128/mr.56.1.100-122.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chapon C., Kolb A. Action of CAP on the malT promoter in vitro. J Bacteriol. 1983 Dec;156(3):1135–1143. doi: 10.1128/jb.156.3.1135-1143.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donnelly C. E., Reznikoff W. S. Mutations in the lac P2 promoter. J Bacteriol. 1987 May;169(5):1812–1817. doi: 10.1128/jb.169.5.1812-1817.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eschenlauer A. C., Reznikoff W. S. Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription. J Bacteriol. 1991 Aug;173(16):5024–5029. doi: 10.1128/jb.173.16.5024-5029.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Herbert M., Kolb A., Buc H. Overlapping promoters and their control in Escherichia coli: the gal case. Proc Natl Acad Sci U S A. 1986 May;83(9):2807–2811. doi: 10.1073/pnas.83.9.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem. 1989;58:913–949. doi: 10.1146/annurev.bi.58.070189.004405. [DOI] [PubMed] [Google Scholar]
  19. Macaluso A., Best E. A., Bender R. A. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J Bacteriol. 1990 Dec;172(12):7249–7255. doi: 10.1128/jb.172.12.7249-7255.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Malan T. P., Kolb A., Buc H., McClure W. R. Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter. J Mol Biol. 1984 Dec 25;180(4):881–909. doi: 10.1016/0022-2836(84)90262-6. [DOI] [PubMed] [Google Scholar]
  21. Malan T. P., McClure W. R. Dual promoter control of the Escherichia coli lactose operon. Cell. 1984 Nov;39(1):173–180. doi: 10.1016/0092-8674(84)90203-4. [DOI] [PubMed] [Google Scholar]
  22. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Menendez M., Kolb A., Buc H. A new target for CRP action at the malT promoter. EMBO J. 1987 Dec 20;6(13):4227–4234. doi: 10.1002/j.1460-2075.1987.tb02771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moore J., Kantorow M., Vanderzwaag D., McKenney K. Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation. J Bacteriol. 1992 Dec;174(24):8030–8035. doi: 10.1128/jb.174.24.8030-8035.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Movva R. N., Green P., Nakamura K., Inouye M. Interaction of cAMP receptor protein with the ompA gene, a gene for a major outer membrane protein of Escherichia coli. FEBS Lett. 1981 Jun 15;128(2):186–190. doi: 10.1016/0014-5793(81)80077-4. [DOI] [PubMed] [Google Scholar]
  27. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nieuwkoop A. J., Baldauf S. A., Hudspeth M. E., Bender R. A. Bidirectional promoter in the hut(P) region of the histidine utilization (hut) operons from Klebsiella aerogenes. J Bacteriol. 1988 May;170(5):2240–2246. doi: 10.1128/jb.170.5.2240-2246.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nieuwkoop A. J., Bender R. A. RNA polymerase as a repressor of transcription in the hut(P) region of mutant Klebsiella aerogenes histidine utilization operons. J Bacteriol. 1988 Oct;170(10):4986–4990. doi: 10.1128/jb.170.10.4986-4990.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nieuwkoop A. J., Boylan S. A., Bender R. A. Regulation of hutUH operon expression by the catabolite gene activator protein-cyclic AMP complex in Klebsiella aerogenes. J Bacteriol. 1984 Sep;159(3):934–939. doi: 10.1128/jb.159.3.934-939.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Osuna R., Bender R. A. Klebsiella aerogenes catabolite gene activator protein and the gene encoding it (crp). J Bacteriol. 1991 Oct;173(20):6626–6631. doi: 10.1128/jb.173.20.6626-6631.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Osuna R., Boylan S. A., Bender R. A. In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes. J Bacteriol. 1991 Jan;173(1):116–123. doi: 10.1128/jb.173.1.116-123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Osuna R., Finkel S. E., Johnson R. C. Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision. EMBO J. 1991 Jun;10(6):1593–1603. doi: 10.1002/j.1460-2075.1991.tb07680.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Osuna R., Schwacha A., Bender R. A. Identification of the hutUH operator (hutUo) from Klebsiella aerogenes by DNA deletion analysis. J Bacteriol. 1994 Sep;176(17):5525–5529. doi: 10.1128/jb.176.17.5525-5529.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Queen C., Rosenberg M. A promoter of pBR322 activated by cAMP receptor protein. Nucleic Acids Res. 1981 Jul 24;9(14):3365–3377. doi: 10.1093/nar/9.14.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ren Y. L., Garges S., Adhya S., Krakow J. S. Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4138–4142. doi: 10.1073/pnas.85.12.4138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reznikoff W. S. Catabolite gene activator protein activation of lac transcription. J Bacteriol. 1992 Feb;174(3):655–658. doi: 10.1128/jb.174.3.655-658.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ross W., Gosink K. K., Salomon J., Igarashi K., Zou C., Ishihama A., Severinov K., Gourse R. L. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. doi: 10.1126/science.8248780. [DOI] [PubMed] [Google Scholar]
  39. Schultz S. C., Shields G. C., Steitz T. A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science. 1991 Aug 30;253(5023):1001–1007. doi: 10.1126/science.1653449. [DOI] [PubMed] [Google Scholar]
  40. Schwacha A., Bender R. A. The product of the Klebsiella aerogenes nac (nitrogen assimilation control) gene is sufficient for activation of the hut operons and repression of the gdh operon. J Bacteriol. 1993 Apr;175(7):2116–2124. doi: 10.1128/jb.175.7.2116-2124.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shanblatt S. H., Revzin A. Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1594–1598. doi: 10.1073/pnas.80.6.1594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smith G. R., Halpern Y. S., Magasanik B. Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. 4-imidazolone-5-propionate amidohydrolase and N-formimino-L-glutamate formiminohydrolase. J Biol Chem. 1971 May 25;246(10):3320–3329. [PubMed] [Google Scholar]
  43. Spassky A., Busby S., Buc H. On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. EMBO J. 1984 Jan;3(1):43–50. doi: 10.1002/j.1460-2075.1984.tb01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  45. Weber I. T., Steitz T. A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J Mol Biol. 1987 Nov 20;198(2):311–326. doi: 10.1016/0022-2836(87)90315-9. [DOI] [PubMed] [Google Scholar]
  46. Yu X. M., Reznikoff W. S. Deletion analysis of the Escherichia coli lactose promoter P2. Nucleic Acids Res. 1985 Apr 11;13(7):2457–2468. doi: 10.1093/nar/13.7.2457. [DOI] [PMC free article] [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]

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

RESOURCES