Abstract
Multisubunit RNA polymerases are targets of sophisticated signal transduction pathways that link environmental or temporal cues to changes in gene expression. Here we show that the sigma 54 protein (σ54), responsible for promoter specific binding by bacterial RNA polymerase, undergoes a nucleotide hydrolysis dependent isomerization on DNA. Changes in protein structure are evident. The isomerization has all the known requirements of σ54-dependent transcription, including a dependence on enhancer binding activator proteins and occurs independently of the core RNA polymerase. We suggest that activator driven changes in σ54 conformation trigger the conversion of a transcriptionally silent RNA polymerase conformation to one able to interact productively with template DNA. Our results illustrate the types of changes that must occur for multisubunit complexes to manipulate DNA, and show that transcription activators can remodel key nucleoprotein structures to achieve direct activation of transcription.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gross, C.A. et al. The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb. Symp. Quant. Biol. 63, 141– 155 (1998).
Sasse-Dwight, S. & Gralla, J.D. Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor σ54. Cell 62, 945– 954 (1990).
Reitzer, L.J. & Magasanik, B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter . Cell 45, 785–792 (1986).
Popham, D.L., Szeto, D., Keener, J. & Kustu, S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science 243, 629–635 ( 1989).
Wedel, A. & Kustu, S. The bacterial enhancer-binding protein NtrC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation. Genes Dev. 9, 2042– 2052 (1995).
Cannon, W., Gallegos, M.T., Casaz, P. & Buck, M. Amino terminal sequences of σN (σ54) inhibit RNA polymerase isomerisation. Genes Dev. 13, 357– 370 (1999).
Neuwald, A.F., Aravind, L., Spouge, J.L. & Koonin, E.V. AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27– 43 (1999).
Wang, J.T., Syed, A. & Gralla, J.D. Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants. Proc. Natl. Acad. Sci. USA 94, 9538–9543 ( 1997).
Gallegos, M.T. & Buck, M. Sequences in region I required for binding to early melted DNA and their involvement in sigma-DNA isomerisation . J. Mol. Biol. 297, 849– 859 (2000).
Hsieh, M. & Gralla, J.D. Analysis of the N-terminal leucine heptad and hexad repeats of sigma 54. J. Mol. Biol. 239, 15–24 (1994).
Hsieh, M., Tintut, Y. & Gralla, J.D. Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54. J. Biol. Chem. 269, 373–378 (1994).
Syed, A. & Gralla, J.D. Identification of an N-terminal region of sigma 54 required for enhancer responsiveness. J. Bacteriol. 180, 5619–5625 ( 1998).
Merrick, M.J. In a class of its own-the RNA polymerase sigma factor sigma 54 (sigma N). Mol. Microbiol. 10, 903–909 (1993).
Morris, L., Cannon, W., Claverie-Martin, F., Austin, S. & Buck, M. DNA distortion and nucleation of local DNA unwinding within sigma-54 (σN) holoenzyme closed promoter complexes. J. Biol. Chem. 269, 11563–11571 (1994).
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 270, 992–994 ( 1995).
Wang, J.T. & Gralla, J.D. The transcription initiation pathway of sigma 54 mutants that bypass the enhancer protein requirement. Implications for the mechanism of activation. J. Biol. Chem. 271 , 32707–32713 (1996).
Gallegos, M.T. & Buck, M. Sequences in σN determining holoenzyme formation and properties. J. Mol. Biol. 288, 539–553 ( 1999).
Casaz, P. & Buck, M. Probing the assembly of transcription initiation complexes through changes in σN protease sensitivity . Proc. Natl. Acad. Sci. USA 94, 12145– 12150 (1997).
Casaz, P. & Buck, M. Region I modifies DNA binding domain conformation of sigma 54 holoenzyme. J. Mol. Biol. 285, 507–514 (1999).
Chaney, M. & Buck, M. The sigma 54 DNA-binding domain includes a determinant of enhancer responsiveness. Mol. Microbiol. 33, 1200–1209 (1999).
Guo, Y., Wang, L. & Gralla, J.D. A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor. EMBO J. 18, 3736–3745 (1999).
Wang, L. & Gralla, J.D. Multiple in vivo roles for the −12-region elements of sigma 54 promoters. J. Bacteriol. 180, 5626–5631 (1998).
Buck, M. & Cannon, W. Specific binding of the transcription factor sigma-54 to promoter DNA. Nature 358, 422 –424 (1992).
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 67, 155–167 (1991).
González, V., Olvera, L., Soberón, X. & Morett, E. In vivo studies on the positive control function of NifA: a conserved hydrophobic amino acid patch at the central domain involved in transcriptional activation. Mol. Microbiol. 28, 55– 67 (1998).
Wang, Y.K. & Hoover, T.R. Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation. J. Bacteriol. 179, 5812– 5819 (1997).
Oguiza, J.A., Gallegos, M.T., Chaney, M.K., Cannon, W.V. & Buck, M. Involvement of the σN DNA-binding domain in open complex formation. Mol. Microbiol. 33 , 873–885 (1999).
Jovanovic, G., Rakonjac, J. & Model, P. In vivo and in vitro activities of the Escherichia coli σ54 transcription activator, PspF, and its DNA-binding mutant, PspFΔHTH . J. Mol. Biol. 285, 469– 483 (1999).
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. 176, 3460–3465 ( 1994).
Hunt, T.P. & Magasanik, B. Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL. Proc. Natl. Acad. Sci. USA 82, 8453–8457 ( 1985).
Klose, K.E., North, A.K., Stedman, K.M. & Kustu, S. The major dimerization determinants of the nitrogen regulatory protein NtrC from enteric bacteria lie in its carboxy-terminal domain. J. Mol. Biol. 241 , 233–245 (1994).
Rombel, I., North, A., Hwang, I., Wyman, C. & Kustu, S. The bacterial enhancer-binding protein NtrC as a molecular machine. Cold Spring Harb. Symp. Quant. Biol. 63, 157–166 (1998).
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. USA 92, 9702–9706 ( 1995).
Gallegos, M.T., Cannon, W. & Buck, M. Functions of the σ54 region I in trans and implications for transcription activation. J. Biol. Chem. 274, 25285–25290 (1999).
Buckle, M., Pemberton, I.K., Jacquet, M.A. & Buc, H. The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase. J. Mol. Biol. 285, 955–964 (1999).
Reinberg et al. The RNA polymerase II general transcription factors: past, present and future. Cold Spring Harb. Symp. Quant. Biol. 63, 83– 103 (1998).
Fu, J. et al. Yeast RNA polymerase II at 5 Å resolution. Cell 98, 799–810 (1999).
Zhang, G. et al. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell 98, 811– 824 (1999).
Tinker-Kulberg, R.L., Fu, T.J., Geiduschek, E.P. & Kassavetis, G.A. A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence. EMBO J. 15, 5032–5039 (1996).
Cannon, W. et al. Core RNA polymerase and promoter DNA interactions of purified domains of sigma N: bipartite functions. J. Mol. Biol. 248, 781–803 (1995).
Acknowledgements
Work was supported by a Wellcome trust project grant to M.B. M.T.G. was supported by a CEC Marie Curie fellowship. We thank S. Kustu, L.J. Reitzer, R. Wassem and S.R. Wigneshweraraj for purified activator proteins, E. Morett for the pspF mutants, D. Studholme for valuable comments on the manuscript and P. Geiduschek for his constructive comments on the early work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cannon, W., Gallegos, MT. & Buck, M. Isomerization of a binary sigma–promoter DNA complex by transcription activators. Nat Struct Mol Biol 7, 594–601 (2000). https://doi.org/10.1038/76830
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/76830
This article is cited by
-
Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells
Nature Communications (2013)
-
A non-haem iron centre in the transcription factor NorR senses nitric oxide
Nature (2005)
-
Reorganisation of an RNA polymerase–promoter DNA complex for DNA melting
The EMBO Journal (2004)
-
Regulated communication between the upstream face of RNA polymerase and the β′ subunit jaw domain
The EMBO Journal (2004)