Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Feb 15;330(Pt 1):413–420. doi: 10.1042/bj3300413

Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

G S Lloyd 1, S J Busby 1, N J Savery 1
PMCID: PMC1219155  PMID: 9461538

Abstract

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed.

Full Text

The Full Text of this article is available as a PDF (873.8 KB).

Selected References

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

  1. Attey A., Belyaeva T., Savery N., Hoggett J., Fujita N., Ishihama A., Busby S. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter. Nucleic Acids Res. 1994 Oct 25;22(21):4375–4380. doi: 10.1093/nar/22.21.4375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Belyaeva T. A., Bown J. A., Fujita N., Ishihama A., Busby S. J. Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region. Nucleic Acids Res. 1996 Jun 15;24(12):2242–2251. doi: 10.1093/nar/24.12.2243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Busby S., Aiba H., de Crombrugghe B. Mutations in the Escherichia coli operon that define two promoters and the binding site of the cyclic AMP receptor protein. J Mol Biol. 1982 Jan 15;154(2):211–227. doi: 10.1016/0022-2836(82)90061-4. [DOI] [PubMed] [Google Scholar]
  5. Busby S., Ebright R. H. Transcription activation at class II CAP-dependent promoters. Mol Microbiol. 1997 Mar;23(5):853–859. doi: 10.1046/j.1365-2958.1997.2771641.x. [DOI] [PubMed] [Google Scholar]
  6. Busby S., West D., Lawes M., Webster C., Ishihama A., Kolb A. Transcription activation by the Escherichia coli cyclic AMP receptor protein. Receptors bound in tandem at promoters can interact synergistically. J Mol Biol. 1994 Aug 19;241(3):341–352. doi: 10.1006/jmbi.1994.1511. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  8. Ebright R. H., Busby S. The Escherichia coli RNA polymerase alpha subunit: structure and function. Curr Opin Genet Dev. 1995 Apr;5(2):197–203. doi: 10.1016/0959-437x(95)80008-5. [DOI] [PubMed] [Google Scholar]
  9. Eichenberger P., Déthiollaz S., Fujita N., Ishihama A., Geiselmann J. Influence of the location of the cAMP receptor protein binding site on the geometry of a transcriptional activation complex in Escherichia coli. Biochemistry. 1996 Dec 3;35(48):15302–15312. doi: 10.1021/bi961377d. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Ghosaini L. R., Brown A. M., Sturtevant J. M. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry. 1988 Jul 12;27(14):5257–5261. doi: 10.1021/bi00414a046. [DOI] [PubMed] [Google Scholar]
  12. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kolb A., Spassky A., Chapon C., Blazy B., Buc H. On the different binding affinities of CRP at the lac, gal and malT promoter regions. Nucleic Acids Res. 1983 Nov 25;11(22):7833–7852. doi: 10.1093/nar/11.22.7833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lodge J., Fear J., Busby S., Gunasekaran P., Kamini N. R. Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiol Lett. 1992 Aug 15;74(2-3):271–276. doi: 10.1016/0378-1097(92)90441-p. [DOI] [PubMed] [Google Scholar]
  17. Murakami K., Kimura M., Owens J. T., Meares C. F., Ishihama A. The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1709–1714. doi: 10.1073/pnas.94.5.1709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Niu W., Zhou Y., Dong Q., Ebright Y. W., Ebright R. H. Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis. J Mol Biol. 1994 Nov 4;243(4):595–602. doi: 10.1016/0022-2836(94)90034-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Savery N. J., Rhodius V. A., Wing H. J., Busby S. J. Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit. Biochem J. 1995 Jul 1;309(Pt 1):77–83. doi: 10.1042/bj3090077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tang H., Severinov K., Goldfarb A., Ebright R. H. Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4902–4906. doi: 10.1073/pnas.92.11.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zhou Y., Merkel T. J., Ebright R. H. Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). II. Role at Class I and class II CAP-dependent promoters. J Mol Biol. 1994 Nov 4;243(4):603–610. doi: 10.1016/0022-2836(94)90035-3. [DOI] [PubMed] [Google Scholar]
  24. Zhou Y., Pendergrast P. S., Bell A., Williams R., Busby S., Ebright R. H. The functional subunit of a dimeric transcription activator protein depends on promoter architecture. EMBO J. 1994 Oct 3;13(19):4549–4557. doi: 10.1002/j.1460-2075.1994.tb06776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES