Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jun 15;17(12):3439–3447. doi: 10.1093/emboj/17.12.3439

Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.

N J Savery 1, G S Lloyd 1, M Kainz 1, T Gaal 1, W Ross 1, R H Ebright 1, R L Gourse 1, S J Busby 1
PMCID: PMC1170680  PMID: 9628879

Abstract

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

Full Text

The Full Text of this article is available as a PDF (624.0 KB).

Selected References

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

  1. Artsimovitch I., Murakami K., Ishihama A., Howe M. M. Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase. J Biol Chem. 1996 Dec 13;271(50):32343–32348. doi: 10.1074/jbc.271.50.32343. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Busby S., Kotlarz D., Buc H. Deletion mutagenesis of the Escherichia coli galactose operon promoter region. J Mol Biol. 1983 Jun 25;167(2):259–274. doi: 10.1016/s0022-2836(83)80335-0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  11. Dove S. L., Joung J. K., Hochschild A. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature. 1997 Apr 10;386(6625):627–630. doi: 10.1038/386627a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gaal T., Ross W., Blatter E. E., Tang H., Jia X., Krishnan V. V., Assa-Munt N., Ebright R. H., Gourse R. L. DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture. Genes Dev. 1996 Jan 1;10(1):16–26. doi: 10.1101/gad.10.1.16. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Jeon Y. H., Negishi T., Shirakawa M., Yamazaki T., Fujita N., Ishihama A., Kyogoku Y. Solution structure of the activator contact domain of the RNA polymerase alpha subunit. Science. 1995 Dec 1;270(5241):1495–1497. doi: 10.1126/science.270.5241.1495. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kolb A., Kotlarz D., Kusano S., Ishihama A. Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation. Nucleic Acids Res. 1995 Mar 11;23(5):819–826. doi: 10.1093/nar/23.5.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Lombardo M. J., Bagga D., Miller C. G. Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium. J Bacteriol. 1991 Dec;173(23):7511–7518. doi: 10.1128/jb.173.23.7511-7518.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murakami K., Fujita N., Ishihama A. Transcription factor recognition surface on the RNA polymerase alpha subunit is involved in contact with the DNA enhancer element. EMBO J. 1996 Aug 15;15(16):4358–4367. [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Niu W., Kim Y., Tau G., Heyduk T., Ebright R. H. Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell. 1996 Dec 13;87(6):1123–1134. doi: 10.1016/s0092-8674(00)81806-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Rao L., Ross W., Appleman J. A., Gaal T., Leirmo S., Schlax P. J., Record M. T., Jr, Gourse R. L. Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength. J Mol Biol. 1994 Feb 4;235(5):1421–1435. doi: 10.1006/jmbi.1994.1098. [DOI] [PubMed] [Google Scholar]
  27. Rhodius V. A., Busby S. J. Positive activation of gene expression. Curr Opin Microbiol. 1998 Apr;1(2):152–159. doi: 10.1016/s1369-5274(98)80005-2. [DOI] [PubMed] [Google Scholar]
  28. Rhodius V. A., West D. M., Webster C. L., Busby S. J., Savery N. J. Transcription activation at class II CRP-dependent promoters: the role of different activating regions. Nucleic Acids Res. 1997 Jan 15;25(2):326–332. doi: 10.1093/nar/25.2.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Severinov K., Soushko M., Goldfarb A., Nikiforov V. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase. J Biol Chem. 1993 Jul 15;268(20):14820–14825. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Tang H., Severinov K., Goldfarb A., Fenyo D., Chait B., Ebright R. H. Location, structure, and function of the target of a transcriptional activator protein. Genes Dev. 1994 Dec 15;8(24):3058–3067. doi: 10.1101/gad.8.24.3058. [DOI] [PubMed] [Google Scholar]
  36. West D., Williams R., Rhodius V., Bell A., Sharma N., Zou C., Fujita N., Ishihama A., Busby S. Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at class II promoters. Mol Microbiol. 1993 Nov;10(4):789–797. doi: 10.1111/j.1365-2958.1993.tb00949.x. [DOI] [PubMed] [Google Scholar]
  37. Wood L. F., Tszine N. Y., Christie G. E. Activation of P2 late transcription by P2 Ogr protein requires a discrete contact site on the C terminus of the alpha subunit of Escherichia coli RNA polymerase. J Mol Biol. 1997 Nov 21;274(1):1–7. doi: 10.1006/jmbi.1997.1390. [DOI] [PubMed] [Google Scholar]
  38. Zalenskaya K., Lee J., Gujuluva C. N., Shin Y. K., Slutsky M., Goldfarb A. Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products. Gene. 1990 Apr 30;89(1):7–12. doi: 10.1016/0378-1119(90)90199-2. [DOI] [PubMed] [Google Scholar]
  39. Zhou Y. H., Zhang X. P., Ebright R. H. Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res. 1991 Nov 11;19(21):6052–6052. doi: 10.1093/nar/19.21.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhou Y., Busby S., Ebright R. H. Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers. Cell. 1993 Apr 23;73(2):375–379. doi: 10.1016/0092-8674(93)90236-j. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Zou C., Fujita N., Igarashi K., Ishihama A. Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase. Mol Microbiol. 1992 Sep;6(18):2599–2605. doi: 10.1111/j.1365-2958.1992.tb01437.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES