Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Sep;178(17):5182–5187. doi: 10.1128/jb.178.17.5182-5187.1996

A positive control mutant of the transcription activator protein FIS.

K K Gosink 1, T Gaal 1, A J Bokal 4th 1, R L Gourse 1
PMCID: PMC178315  PMID: 8752336

Abstract

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.

Full Text

The Full Text of this article is available as a PDF (682.0 KB).

Selected References

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

  1. Bartlett M. S., Gourse R. L. Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli. J Bacteriol. 1994 Sep;176(17):5560–5564. doi: 10.1128/jb.176.17.5560-5564.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cole J. R., Olsson C. L., Hershey J. W., Grunberg-Manago M., Nomura M. Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2. J Mol Biol. 1987 Dec 5;198(3):383–392. doi: 10.1016/0022-2836(87)90288-9. [DOI] [PubMed] [Google Scholar]
  4. Eschenlauer A. C., Reznikoff W. S. Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription. J Bacteriol. 1991 Aug;173(16):5024–5029. doi: 10.1128/jb.173.16.5024-5029.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Gourse R. L., Gaal T., Bartlett M. S., Appleman J. A., Ross W. rRNA transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli. Annu Rev Microbiol. 1996;50:645–677. doi: 10.1146/annurev.micro.50.1.645. [DOI] [PubMed] [Google Scholar]
  10. Gourse R. L., Takebe Y., Sharrock R. A., Nomura M. Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1069–1073. doi: 10.1073/pnas.82.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haffter P., Bickle T. A. Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin. J Mol Biol. 1987 Dec 20;198(4):579–587. doi: 10.1016/0022-2836(87)90201-4. [DOI] [PubMed] [Google Scholar]
  12. Hochschild A., Irwin N., Ptashne M. Repressor structure and the mechanism of positive control. Cell. 1983 Feb;32(2):319–325. doi: 10.1016/0092-8674(83)90451-8. [DOI] [PubMed] [Google Scholar]
  13. Jinks-Robertson S., Gourse R. L., Nomura M. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell. 1983 Jul;33(3):865–876. doi: 10.1016/0092-8674(83)90029-6. [DOI] [PubMed] [Google Scholar]
  14. Johnson R. C., Bruist M. F., Simon M. I. Host protein requirements for in vitro site-specific DNA inversion. Cell. 1986 Aug 15;46(4):531–539. doi: 10.1016/0092-8674(86)90878-0. [DOI] [PubMed] [Google Scholar]
  15. Johnson R. C., Simon M. I. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer. Cell. 1985 Jul;41(3):781–791. doi: 10.1016/s0092-8674(85)80059-3. [DOI] [PubMed] [Google Scholar]
  16. Josaitis C. A., Gaal T., Gourse R. L. Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1117–1121. doi: 10.1073/pnas.92.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  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. Nilsson L., Emilsson V. Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli. J Biol Chem. 1994 Apr 1;269(13):9460–9465. [PubMed] [Google Scholar]
  24. 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]
  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. 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]
  27. 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]
  28. 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]
  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. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Yamagishi M., de Boer H. A., Nomura M. Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation. J Mol Biol. 1987 Dec 5;198(3):547–550. doi: 10.1016/0022-2836(87)90299-3. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  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., Zhang X., Ebright R. H. Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6081–6085. doi: 10.1073/pnas.90.13.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES