Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Sep 1;25(17):3478–3485. doi: 10.1093/nar/25.17.3478

Nucleoprotein complex formation by the enhancer binding protein nifA.

X Y Wang 1, A Kolb 1, W Cannon 1, M Buck 1
PMCID: PMC146925  PMID: 9254707

Abstract

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

Full Text

The Full Text of this article is available as a PDF (190.7 KB).

Selected References

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

  1. Austin S., Buck M., Cannon W., Eydmann T., Dixon R. Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL. J Bacteriol. 1994 Jun;176(12):3460–3465. doi: 10.1128/jb.176.12.3460-3465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Austin S., Dixon R. The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J. 1992 Jun;11(6):2219–2228. doi: 10.1002/j.1460-2075.1992.tb05281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Austin S., Kundrot C., Dixon R. Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function. Nucleic Acids Res. 1991 May 11;19(9):2281–2287. doi: 10.1093/nar/19.9.2281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger D. K., Narberhaus F., Kustu S. The isolated catalytic domain of NIFA, a bacterial enhancer-binding protein, activates transcription in vitro: activation is inhibited by NIFL. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):103–107. doi: 10.1073/pnas.91.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buck M., Cannon W. Activator-independent formation of a closed complex between sigma 54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae. Mol Microbiol. 1992 Jun;6(12):1625–1630. doi: 10.1111/j.1365-2958.1992.tb00887.x. [DOI] [PubMed] [Google Scholar]
  6. Buck M., Cannon W. Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences. Nucleic Acids Res. 1989 Apr 11;17(7):2597–2612. doi: 10.1093/nar/17.7.2597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buck M., Cannon W. Specific binding of the transcription factor sigma-54 to promoter DNA. Nature. 1992 Jul 30;358(6385):422–424. doi: 10.1038/358422a0. [DOI] [PubMed] [Google Scholar]
  8. Buckle M., Geiselmann J., Kolb A., Buc H. Protein-DNA cross-linking at the lac promoter. Nucleic Acids Res. 1991 Feb 25;19(4):833–840. doi: 10.1093/nar/19.4.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cannon W., Austin S., Moore M., Buck M. Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes. Nucleic Acids Res. 1995 Feb 11;23(3):351–356. doi: 10.1093/nar/23.3.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cannon W., Claverie-Martin F., Austin S., Buck M. Core RNA polymerase assists binding of the transcription factor sigma 54 to promoter DNA. Mol Microbiol. 1993 Apr;8(2):287–298. doi: 10.1111/j.1365-2958.1993.tb01573.x. [DOI] [PubMed] [Google Scholar]
  11. Cannon W., Missailidis S., Smith C., Cottier A., Austin S., Moore M., Buck M. Core RNA polymerase and promoter DNA interactions of purified domains of sigma N: bipartite functions. J Mol Biol. 1995 May 12;248(4):781–803. doi: 10.1006/jmbi.1995.0260. [DOI] [PubMed] [Google Scholar]
  12. Charlton W., Cannon W., Buck M. The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter. Mol Microbiol. 1993 Mar;7(6):1007–1021. doi: 10.1111/j.1365-2958.1993.tb01192.x. [DOI] [PubMed] [Google Scholar]
  13. Engelhorn M., Boccard F., Murtin C., Prentki P., Geiselmann J. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites. Nucleic Acids Res. 1995 Sep 11;23(17):2959–2965. [PubMed] [Google Scholar]
  14. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee J. H., Hoover T. R. Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9702–9706. doi: 10.1073/pnas.92.21.9702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee J. H., Scholl D., Nixon B. T., Hoover T. R. Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator. J Biol Chem. 1994 Aug 12;269(32):20401–20409. [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. Merrick M. J. In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N). Mol Microbiol. 1993 Dec;10(5):903–909. doi: 10.1111/j.1365-2958.1993.tb00961.x. [DOI] [PubMed] [Google Scholar]
  19. Morett E., Buck M. In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. J Mol Biol. 1989 Nov 5;210(1):65–77. doi: 10.1016/0022-2836(89)90291-x. [DOI] [PubMed] [Google Scholar]
  20. Morett E., Segovia L. The sigma 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol. 1993 Oct;175(19):6067–6074. doi: 10.1128/jb.175.19.6067-6074.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morris L., Cannon W., Claverie-Martin F., Austin S., Buck M. DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes. J Biol Chem. 1994 Apr 15;269(15):11563–11571. [PubMed] [Google Scholar]
  22. North A. K., Klose K. E., Stedman K. M., Kustu S. Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: the puzzle of nitrogen regulatory protein C. J Bacteriol. 1993 Jul;175(14):4267–4273. doi: 10.1128/jb.175.14.4267-4273.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Popham D., Keener J., Kustu S. Purification of the alternative sigma factor, sigma 54, from Salmonella typhimurium and characterization of sigma 54-holoenzyme. J Biol Chem. 1991 Oct 15;266(29):19510–19518. [PubMed] [Google Scholar]
  24. Santero E., Hoover T. R., North A. K., Berger D. K., Porter S. C., Kustu S. Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter. J Mol Biol. 1992 Oct 5;227(3):602–620. doi: 10.1016/0022-2836(92)90211-2. [DOI] [PubMed] [Google Scholar]
  25. Su W., Porter S., Kustu S., Echols H. DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5504–5508. doi: 10.1073/pnas.87.14.5504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wang J. T., Syed A., Hsieh M., Gralla J. D. Converting Escherichia coli RNA polymerase into an enhancer-responsive enzyme: role of an NH2-terminal leucine patch in sigma 54. Science. 1995 Nov 10;270(5238):992–994. doi: 10.1126/science.270.5238.992. [DOI] [PubMed] [Google Scholar]
  27. Weiss D. S., Batut J., Klose K. E., Keener J., Kustu S. The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription. Cell. 1991 Oct 4;67(1):155–167. doi: 10.1016/0092-8674(91)90579-n. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES