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. 1997 Jan 2;16(1):154–162. doi: 10.1093/emboj/16.1.154

Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter.

A J Bokal 1, W Ross 1, T Gaal 1, R C Johnson 1, R L Gourse 1
PMCID: PMC1169622  PMID: 9009276

Abstract

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.

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Selected References

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  1. Ball C. A., Johnson R. C. Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein. J Bacteriol. 1991 Jul;173(13):4027–4031. doi: 10.1128/jb.173.13.4027-4031.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bokal A. J., 4th, Ross W., Gourse R. L. The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter. J Mol Biol. 1995 Jan 20;245(3):197–207. doi: 10.1006/jmbi.1994.0016. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Finkel S. E., Johnson R. C. The Fis protein: it's not just for DNA inversion anymore. Mol Microbiol. 1992 Nov;6(22):3257–3265. doi: 10.1111/j.1365-2958.1992.tb02193.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  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. 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]
  12. Gosink K. K., Gaal T., Bokal A. J., 4th, Gourse R. L. A positive control mutant of the transcription activator protein FIS. J Bacteriol. 1996 Sep;178(17):5182–5187. doi: 10.1128/jb.178.17.5182-5187.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gosink K. K., Ross W., Leirmo S., Osuna R., Finkel S. E., Johnson R. C., Gourse R. L. DNA binding and bending are necessary but not sufficient for Fis-dependent activation of rrnB P1. J Bacteriol. 1993 Mar;175(6):1580–1589. doi: 10.1128/jb.175.6.1580-1589.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kahmann R., Rudt F., Koch C., Mertens G. G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor. Cell. 1985 Jul;41(3):771–780. doi: 10.1016/s0092-8674(85)80058-1. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Pan C. Q., Feng J. A., Finkel S. E., Landgraf R., Sigman D., Johnson R. C. Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1721–1725. doi: 10.1073/pnas.91.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Savery N., Rhodius V., Busby S. Protein-protein interactions during transcription activation: the case of the Escherichia coli cyclic AMP receptor protein. Philos Trans R Soc Lond B Biol Sci. 1996 Apr 29;351(1339):543–550. doi: 10.1098/rstb.1996.0053. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Tao K., Zou C., Fujita N., Ishihama A. Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit. J Bacteriol. 1995 Dec;177(23):6740–6744. doi: 10.1128/jb.177.23.6740-6744.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Weinreich M. D., Reznikoff W. S. Fis plays a role in Tn5 and IS50 transposition. J Bacteriol. 1992 Jul;174(14):4530–4537. doi: 10.1128/jb.174.14.4530-4537.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Yuan H. S., Finkel S. E., Feng J. A., Kaczor-Grzeskowiak M., Johnson R. C., Dickerson R. E. The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9558–9562. doi: 10.1073/pnas.88.21.9558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zacharias M., Göringer H. U., Wagner R. Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter. Biochemistry. 1992 Mar 10;31(9):2621–2628. doi: 10.1021/bi00124a024. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Zou C., Fujita N., Ishihama A. Asymmetric arrangement of two alpha subunits within Escherichia coli RNA polymerase. Involvement of one alpha subunit in contact with cAMP receptor protein. J Mol Biol. 1994 Mar 11;236(5):1283–1288. doi: 10.1016/0022-2836(94)90057-4. [DOI] [PubMed] [Google Scholar]

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