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. 1999 Feb 1;337(Pt 3):415–423.

Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the alpha subunit of RNA polymerase at class I promoters.

E C Law 1, N J Savery 1, S J Busby 1
PMCID: PMC1219992  PMID: 9895284

Abstract

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Since alphaCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP-alphaCTD interaction and the CRP-UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two alphaCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

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Selected References

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  1. 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]
  2. 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]
  3. Belyaeva T. A., Rhodius V. A., Webster C. L., Busby S. J. Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits. J Mol Biol. 1998 Apr 10;277(4):789–804. doi: 10.1006/jmbi.1998.1666. [DOI] [PubMed] [Google Scholar]
  4. Blatter E. E., Ross W., Tang H., Gourse R. L., Ebright R. H. Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell. 1994 Sep 9;78(5):889–896. doi: 10.1016/s0092-8674(94)90682-3. [DOI] [PubMed] [Google Scholar]
  5. Busby S., Ebright R. H. Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell. 1994 Dec 2;79(5):743–746. doi: 10.1016/0092-8674(94)90063-9. [DOI] [PubMed] [Google Scholar]
  6. Czarniecki D., Noel R. J., Jr, Reznikoff W. S. The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription. J Bacteriol. 1997 Jan;179(2):423–429. doi: 10.1128/jb.179.2.423-429.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  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. 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. Estrem S. T., Gaal T., Ross W., Gourse R. L. Identification of an UP element consensus sequence for bacterial promoters. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9761–9766. doi: 10.1073/pnas.95.17.9761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flatow U., Rajendrakumar G. V., Garges S. Analysis of the spacer DNA between the cyclic AMP receptor protein binding site and the lac promoter. J Bacteriol. 1996 Apr;178(8):2436–2439. doi: 10.1128/jb.178.8.2436-2439.1996. [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. 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]
  14. Jeon Y. H., Yamazaki T., Otomo T., Ishihama A., Kyogoku Y. Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain. J Mol Biol. 1997 Apr 11;267(4):953–962. doi: 10.1006/jmbi.1997.0902. [DOI] [PubMed] [Google Scholar]
  15. Joung J. K., Le L. U., Hochschild A. Synergistic activation of transcription by Escherichia coli cAMP receptor protein. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3083–3087. doi: 10.1073/pnas.90.7.3083. [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. Kolb A., Busby S., Herbert M., Kotlarz D., Buc H. Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters. EMBO J. 1983;2(2):217–222. doi: 10.1002/j.1460-2075.1983.tb01408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kolb A., Igarashi K., Ishihama A., Lavigne M., Buckle M., Buc H. E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter. Nucleic Acids Res. 1993 Jan 25;21(2):319–326. doi: 10.1093/nar/21.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lloyd G. S., Busby S. J., Savery N. J. Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein. Biochem J. 1998 Feb 15;330(Pt 1):413–420. doi: 10.1042/bj3300413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Murakami K., Owens J. T., Belyaeva T. A., Meares C. F., Busby S. J., Ishihama A. Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11274–11278. doi: 10.1073/pnas.94.21.11274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Noel R. J., Jr, Reznikoff W. S. CAP, the -45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli. J Mol Biol. 1998 Sep 25;282(3):495–504. doi: 10.1006/jmbi.1998.2040. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Savery N. J., Lloyd G. S., Kainz M., Gaal T., Ross W., Ebright R. H., Gourse R. L., Busby S. J. Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit. EMBO J. 1998 Jun 15;17(12):3439–3447. doi: 10.1093/emboj/17.12.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]

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