Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 May 15;17(10):2877–2885. doi: 10.1093/emboj/17.10.2877

Regulation of crp transcription by oscillation between distinct nucleoprotein complexes.

G González-Gil 1, R Kahmann 1, G Muskhelishvili 1
PMCID: PMC1170628  PMID: 9582281

Abstract

FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

Full Text

The Full Text of this article is available as a PDF (424.0 KB).

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., Fujimoto S., Ozaki N. Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein. Nucleic Acids Res. 1982 Feb 25;10(4):1345–1361. doi: 10.1093/nar/10.4.1345. [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. Borowiec J. A., Gralla J. D. All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling. J Mol Biol. 1987 May 5;195(1):89–97. doi: 10.1016/0022-2836(87)90329-9. [DOI] [PubMed] [Google Scholar]
  5. Botsford J. L., Drexler M. The cyclic 3',5'-adenosine monophosphate receptor protein and regulation of cyclic 3',5'-adenosine monophosphate synthesis in Escherichia coli. Mol Gen Genet. 1978 Sep 20;165(1):47–56. doi: 10.1007/BF00270375. [DOI] [PubMed] [Google Scholar]
  6. Brikun I., Suziedelis K., Stemmann O., Zhong R., Alikhanian L., Linkova E., Mironov A., Berg D. E. Analysis of CRP-CytR interactions at the Escherichia coli udp promoter. J Bacteriol. 1996 Mar;178(6):1614–1622. doi: 10.1128/jb.178.6.1614-1622.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassler M. R., Grimwade J. E., Leonard A. C. Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin. EMBO J. 1995 Dec 1;14(23):5833–5841. doi: 10.1002/j.1460-2075.1995.tb00271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Claret L., Rouviere-Yaniv J. Regulation of HU alpha and HU beta by CRP and FIS in Escherichia coli. J Mol Biol. 1996 Oct 25;263(2):126–139. doi: 10.1006/jmbi.1996.0564. [DOI] [PubMed] [Google Scholar]
  9. Cossart P., Gicquel-Sanzey B. Cloning and sequence of the crp gene of Escherichia coli K 12. Nucleic Acids Res. 1982 Feb 25;10(4):1363–1378. doi: 10.1093/nar/10.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cossart P., Gicquel-Sanzey B. Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study. J Bacteriol. 1985 Jan;161(1):454–457. doi: 10.1128/jb.161.1.454-457.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli. Nature. 1984 Sep 20;311(5983):232–235. doi: 10.1038/311232a0. [DOI] [PubMed] [Google Scholar]
  12. Emilsson V., Nilsson L. Factor for inversion stimulation-dependent growth rate regulation of serine and threonine tRNA species. J Biol Chem. 1995 Jul 14;270(28):16610–16614. doi: 10.1074/jbc.270.28.16610. [DOI] [PubMed] [Google Scholar]
  13. Falconi M., Brandi A., La Teana A., Gualerzi C. O., Pon C. L. Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression. Mol Microbiol. 1996 Mar;19(5):965–975. doi: 10.1046/j.1365-2958.1996.436961.x. [DOI] [PubMed] [Google Scholar]
  14. Filutowicz M., Ross W., Wild J., Gourse R. L. Involvement of Fis protein in replication of the Escherichia coli chromosome. J Bacteriol. 1992 Jan;174(2):398–407. doi: 10.1128/jb.174.2.398-407.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Friden P., Newman T., Freundlich M. Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6156–6160. doi: 10.1073/pnas.79.20.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Giladi H., Koby S., Gottesman M. E., Oppenheim A. B. Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter. J Mol Biol. 1992 Apr 20;224(4):937–948. doi: 10.1016/0022-2836(92)90461-r. [DOI] [PubMed] [Google Scholar]
  18. Gille H., Egan J. B., Roth A., Messer W. The FIS protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli. Nucleic Acids Res. 1991 Aug 11;19(15):4167–4172. doi: 10.1093/nar/19.15.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. González-Gil G., Bringmann P., Kahmann R. FIS is a regulator of metabolism in Escherichia coli. Mol Microbiol. 1996 Oct;22(1):21–29. doi: 10.1111/j.1365-2958.1996.tb02652.x. [DOI] [PubMed] [Google Scholar]
  20. Hanamura A., Aiba H. A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation. Mol Microbiol. 1992 Sep;6(17):2489–2497. doi: 10.1111/j.1365-2958.1992.tb01425.x. [DOI] [PubMed] [Google Scholar]
  21. Hanamura A., Aiba H. Molecular mechanism of negative autoregulation of Escherichia coli crp gene. Nucleic Acids Res. 1991 Aug 25;19(16):4413–4419. doi: 10.1093/nar/19.16.4413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hiasa H., Marians K. J. Fis cannot support oriC DNA replication in vitro. J Biol Chem. 1994 Oct 7;269(40):24999–25003. [PubMed] [Google Scholar]
  23. Holst B., Søgaard-Andersen L., Pedersen H., Valentin-Hansen P. The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter. EMBO J. 1992 Oct;11(10):3635–3643. doi: 10.1002/j.1460-2075.1992.tb05448.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hübner P., Arber W. Mutational analysis of a prokaryotic recombinational enhancer element with two functions. EMBO J. 1989 Feb;8(2):577–585. doi: 10.1002/j.1460-2075.1989.tb03412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ishizuka H., Hanamura A., Kunimura T., Aiba H. A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli. Mol Microbiol. 1993 Oct;10(2):341–350. doi: 10.1111/j.1365-2958.1993.tb01960.x. [DOI] [PubMed] [Google Scholar]
  26. Jordi B. J., Owen-Hughes T. A., Hulton C. S., Higgins C. F. DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium. EMBO J. 1995 Nov 15;14(22):5690–5700. doi: 10.1002/j.1460-2075.1995.tb00256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koch C., Kahmann R. Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J Biol Chem. 1986 Nov 25;261(33):15673–15678. [PubMed] [Google Scholar]
  28. Koch C., Ninnemann O., Fuss H., Kahmann R. The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding. Nucleic Acids Res. 1991 Nov 11;19(21):5915–5922. doi: 10.1093/nar/19.21.5915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Kostrewa D., Granzin J., Koch C., Choe H. W., Raghunathan S., Wolf W., Labahn J., Kahmann R., Saenger W. Three-dimensional structure of the E. coli DNA-binding protein FIS. Nature. 1991 Jan 10;349(6305):178–180. doi: 10.1038/349178a0. [DOI] [PubMed] [Google Scholar]
  31. Lazarus L. R., Travers A. A. The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth. EMBO J. 1993 Jun;12(6):2483–2494. doi: 10.1002/j.1460-2075.1993.tb05903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee N. L., Gielow W. O., Wallace R. G. Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proc Natl Acad Sci U S A. 1981 Feb;78(2):752–756. doi: 10.1073/pnas.78.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liu-Johnson H. N., Gartenberg M. R., Crothers D. M. The DNA binding domain and bending angle of E. coli CAP protein. Cell. 1986 Dec 26;47(6):995–1005. doi: 10.1016/0092-8674(86)90814-7. [DOI] [PubMed] [Google Scholar]
  34. Majerfeld I. H., Miller D., Spitz E., Rickenberg H. V. Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex. Mol Gen Genet. 1981;181(4):470–475. doi: 10.1007/BF00428738. [DOI] [PubMed] [Google Scholar]
  35. Mallick U., Herrlich P. Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5520–5523. doi: 10.1073/pnas.76.11.5520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
  38. Metzger W., Schickor P., Meier T., Werel W., Heumann H. Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase. J Mol Biol. 1993 Jul 5;232(1):35–49. doi: 10.1006/jmbi.1993.1368. [DOI] [PubMed] [Google Scholar]
  39. Mori K., Aiba H. Evidence for negative control of cya transcription by cAMP and cAMP receptor protein in intact Escherichia coli cells. J Biol Chem. 1985 Nov 25;260(27):14838–14843. [PubMed] [Google Scholar]
  40. Muskhelishvili G., Buckle M., Heumann H., Kahmann R., Travers A. A. FIS activates sequential steps during transcription initiation at a stable RNA promoter. EMBO J. 1997 Jun 16;16(12):3655–3665. doi: 10.1093/emboj/16.12.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Newlands J. T., Josaitis C. A., Ross W., Gourse R. L. Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent. Nucleic Acids Res. 1992 Feb 25;20(4):719–726. doi: 10.1093/nar/20.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nilsson L., Vanet A., Vijgenboom E., Bosch L. The role of FIS in trans activation of stable RNA operons of E. coli. EMBO J. 1990 Mar;9(3):727–734. doi: 10.1002/j.1460-2075.1990.tb08166.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ninnemann O., Koch C., Kahmann R. The E.coli fis promoter is subject to stringent control and autoregulation. EMBO J. 1992 Mar;11(3):1075–1083. doi: 10.1002/j.1460-2075.1992.tb05146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ogden S., Haggerty D., Stoner C. M., Kolodrubetz D., Schleif R. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3346–3350. doi: 10.1073/pnas.77.6.3346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Okamoto K., Freundlich M. Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5000–5004. doi: 10.1073/pnas.83.14.5000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Okamoto K., Hara S., Bhasin R., Freundlich M. Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA. J Bacteriol. 1988 Nov;170(11):5076–5079. doi: 10.1128/jb.170.11.5076-5079.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pan C. Q., Finkel S. E., Cramton S. E., Feng J. A., Sigman D. S., Johnson R. C. Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. J Mol Biol. 1996 Dec 13;264(4):675–695. doi: 10.1006/jmbi.1996.0669. [DOI] [PubMed] [Google Scholar]
  48. Prusiner S., Miller R. E., Valentine R. C. Adenosine 3':5'-cyclic monophosphate control of the enzymes of glutamine metabolism in Escherichia coli. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2922–2926. doi: 10.1073/pnas.69.10.2922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ross W., Thompson J. F., Newlands J. T., Gourse R. L. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. doi: 10.1002/j.1460-2075.1990.tb07586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. SADLER J. R., NOVICK A. THE PROPERTIES OF REPRESSOR AND THE KINETICS OF ITS ACTION. J Mol Biol. 1965 Jun;12:305–327. doi: 10.1016/s0022-2836(65)80255-8. [DOI] [PubMed] [Google Scholar]
  51. Sabourin D., Beckwith J. Deletion of the Escherichia coli crp gene. J Bacteriol. 1975 Apr;122(1):338–340. doi: 10.1128/jb.122.1.338-340.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schneider R., Travers A., Muskhelishvili G. FIS modulates growth phase-dependent topological transitions of DNA in Escherichia coli. Mol Microbiol. 1997 Nov;26(3):519–530. doi: 10.1046/j.1365-2958.1997.5951971.x. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Søgaard-Andersen L., Valentin-Hansen P. Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor. Cell. 1993 Nov 5;75(3):557–566. doi: 10.1016/0092-8674(93)90389-8. [DOI] [PubMed] [Google Scholar]
  55. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Thompson J. F., Moitoso de Vargas L., Koch C., Kahmann R., Landy A. Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway. Cell. 1987 Sep 11;50(6):901–908. doi: 10.1016/0092-8674(87)90516-2. [DOI] [PubMed] [Google Scholar]
  57. Weng M., Makaroff C. A., Zalkin H. Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase. J Biol Chem. 1986 Apr 25;261(12):5568–5574. [PubMed] [Google Scholar]
  58. Wold S., Crooke E., Skarstad K. The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro. Nucleic Acids Res. 1996 Sep 15;24(18):3527–3532. doi: 10.1093/nar/24.18.3527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Xu J., Johnson R. C. Fis activates the RpoS-dependent stationary-phase expression of proP in Escherichia coli. J Bacteriol. 1995 Sep;177(18):5222–5231. doi: 10.1128/jb.177.18.5222-5231.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Xu J., Johnson R. C. Identification of genes negatively regulated by Fis: Fis and RpoS comodulate growth-phase-dependent gene expression in Escherichia coli. J Bacteriol. 1995 Feb;177(4):938–947. doi: 10.1128/jb.177.4.938-947.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Xu J., Johnson R. C. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J Bacteriol. 1995 Jun;177(11):3166–3175. doi: 10.1128/jb.177.11.3166-3175.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES