1887

Abstract

transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the −12 promoter element of the promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30–40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the −12 promoter element of the promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of −12 promoter element recognition and mRNA production.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-11-3081
1999-11-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/11/1453081a.html?itemId=/content/journal/micro/10.1099/00221287-145-11-3081&mimeType=html&fmt=ahah

References

  1. Austin, S. & Dixon, R. A. ( 1992; ). The prokaryotic enhancer binding protein NTRC has ATPase activity which is phosphorylation and DNA dependent. EMBO J 11, 2219-2228.
    [Google Scholar]
  2. Austin, S., Buck, M., Cannon, W., Eydmann, T. & Dixon, R. ( 1994; ). Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL. J Bacteriol 176, 3460-3465.
    [Google Scholar]
  3. Birkmann, A. & Bock, A. ( 1989; ). Characterization of a cis-regulatory element necessary for formamate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli. Mol Microbiol 3, 187-195.[CrossRef]
    [Google Scholar]
  4. Buck, M. & Cannon, W. ( 1992; ). Specific binding of the transcription factor sigma-54 to promoter DNA. Nature 358, 422-424.[CrossRef]
    [Google Scholar]
  5. Cannon, W., Claverie-Martin, F., Austin, S. & Buck, M. ( 1993; ). Core RNA polymerase assists binding of the transcription factor σ54 to promoter DNA. Mol Microbiol 8, 287-298.[CrossRef]
    [Google Scholar]
  6. Cannon, W., Austin, S., Moore, M. & Buck, M. ( 1995a; ). Identification of close contacts between the σN54) protein and promoter DNA in closed promoter complexes. Nucleic Acids Res 23, 351-356.[CrossRef]
    [Google Scholar]
  7. Cannon, W., Missailids, S., Smith, C., Cottier, A., Austin, S., Moore, M. & Buck, M. ( 1995b; ). Core RNA polymerase and promoter DNA interactions of purified domains of σN: bipartite functions. J Mol Biol 248, 781-803.[CrossRef]
    [Google Scholar]
  8. Cannon, W. V., Chaney, M. K., Wang, X.-Y. & Buck, M. ( 1997; ). Two domains within σN54) cooperate for DNA binding. Proc Natl Acad Sci USA 94, 5006-5011.[CrossRef]
    [Google Scholar]
  9. Gross, C. A., Lonetto, M. & Losick, R. ( 1992; ). Bacterial sigma factors. In Transcriptional Regulation, pp. 129-195. Edited by S. L. McKnight & K. R. Yamamoto. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  10. Guo, Y. & Gralla, J. D. ( 1997; ). DNA-binding determinants of sigma 54 as deduced from libraries of mutations. J Bacteriol 179, 1239-1245.
    [Google Scholar]
  11. Hsieh, M. & Gralla, J. D. ( 1994; ). Analysis of the N-terminal leucine heptad and hexad repeats of sigma 54. J Mol Biol 239, 15-24.[CrossRef]
    [Google Scholar]
  12. Hsieh, M., Tintut, Y. & Gralla, J. D. ( 1994; ). Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54. J Biol Chem 269, 373-378.
    [Google Scholar]
  13. Merrick, M. & Chambers, S. ( 1992; ). The helix-turn-helix motif of σ54 is involved in recognition of the −13 promoter region. J Bacteriol 174, 7221-7226.
    [Google Scholar]
  14. Merrick, M. J., Gibbons, J. & Toukdarian, A. ( 1987; ). The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins. Mol Gen Genet 210, 323-330.[CrossRef]
    [Google Scholar]
  15. Morris, L., Cannon, W., Claverie-Martin, F., Austin, S. & Buck, M. ( 1994; ). DNA distortion and nucleation of local DNA unwinding within sigma 54 holoenzyme closed promoter complexes. J Biol Chem 269, 11563-11571.
    [Google Scholar]
  16. Popham, D. L., Szeto, D., Keener, J. & Kustu, S. ( 1989; ). Function of a bacterial activator protein that binds to transcriptional enhancers. Science 243, 629-635.[CrossRef]
    [Google Scholar]
  17. Reitzer, L. J. & Magasanik, B. ( 1986; ). Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 45, 785-792.[CrossRef]
    [Google Scholar]
  18. Sasse-Dwight, S. & Gralla, J. D. ( 1988; ). Probing the Escherichia coli glnALG upstream activation mechanism in vivo. Proc Natl Acad Sci USA 85, 8934-8938.[CrossRef]
    [Google Scholar]
  19. Sasse-Dwight, S. & Gralla, J. D. ( 1990; ). Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor sigma 54. Cell 62, 945-954.[CrossRef]
    [Google Scholar]
  20. Smith, G. R., Halpern, Y. S. & Magasanik, B. ( 1971; ). Genetics and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. 4-imidazolone-5-propionate amidohydrolase and N-formimino-l-glutamate formiminohydrolase. J Biol Chem 246, 3320-3329.
    [Google Scholar]
  21. Su, W., Porter, S., Kustu, S. & Echols, H. ( 1990; ). DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc Natl Acad Sci USA 87, 5504-5508.[CrossRef]
    [Google Scholar]
  22. Sundaresan, V., Jones, J. D. G., Ow, D. W. & Ausubel, F. M. ( 1983; ). Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature 301, 728-732.[CrossRef]
    [Google Scholar]
  23. Syed, A. & Gralla, J. D. ( 1997; ). Isolation and properties of enhancer-bypass mutants of sigma 54. Mol Microbiol 23, 987-995.[CrossRef]
    [Google Scholar]
  24. Syed, A. & Gralla, J. D. ( 1998; ). Identification of an N-terminal region of sigma 54 required for enhancer responsiveness. J Bacteriol 180, 5619-5625.
    [Google Scholar]
  25. Taylor, J. W., Ott, J. & Eckstein, F. ( 1985; ). The rapid generation of oligonucleotide-directed mutation at high frequency using phosphothioate-modified DNA. Nucleic Acids Res 13, 8764-8785.
    [Google Scholar]
  26. Taylor, M., Butter, R., Chambers, S., Casimiro, M., Badii, F. & Merrick, M. ( 1996; ). The RpoN-box of the RNA polymerase sigma factor σ54 plays a role in promoter recognition. Mol Microbiol 22, 1045-1054.[CrossRef]
    [Google Scholar]
  27. Tintut, Y. & Gralla, J. D. ( 1995; ). PCR mutagenesis identifies a polymerase-binding sequence of sigma 54 that includes a sigma 70 homology region. J Bacteriol 177, 5818-5825.
    [Google Scholar]
  28. Tintut, Y., Wong, C. W., Jiang, Y., Hsish, M. & Gralla, J. D. ( 1994; ). Polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54. Proc Natl Acad Sci USA 91, 2120-2124.[CrossRef]
    [Google Scholar]
  29. Wang, J., Syed, A., Hsieh, M. & Gralla, J. D. ( 1995; ). Converting Escherichia coli RNA polymerase into an enhancer-responsive enzyme – role of a NH2-terminal leucine patch in sigma 54. Science 270, 992-994.[CrossRef]
    [Google Scholar]
  30. Weiss, D. S., Klose, K. E., Hoover, T. R., North, A. K., Porter, S. C., Wedel, A. B. & Kustu, S. ( 1992; ). Prokaryotic enhancers. In Transcriptional Regulation, pp. 667-694. Edited by S. L. McKnight & K. R. Yamamoto. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  31. Wong, C. W., Tintut, Y. & Gralla, J. D. ( 1994; ). The domain structure of sigma 54 as determined by analysis of a set of deletion mutants. J Mol Biol 236, 81-90.[CrossRef]
    [Google Scholar]
  32. Wyman, C., Rombel, I., North, A. K., Bustamante, C. & Kustu, S. ( 1997; ). Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science 275, 1658-1661.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-11-3081
Loading
/content/journal/micro/10.1099/00221287-145-11-3081
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error