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The σ70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription

Nature Structural & Molecular Biology volume 11, pages 551–557 (2004)Cite this article

Abstract

The σ70 subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation. We provide biochemical evidence that σ70 induces a transcription pause at the lacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript. The σ70-dependent pausing requires an interaction between σ70 and a part of the lac repressor operator sequence resembling a promoter −10 consensus. The polysaccharide heparin triggers the release of σ70 from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between σ70 and core RNAP are weakened. We propose that the binding and retention of σ70 in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble. In addition, we suggest that the σ70 subunit in the elongation complex may provide a target for regulation of gene expression.

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Figure 1: Formation of +17 pause during transcription from the lacUV5 promoter.
Figure 2: Formation of TEC16 during abortive initiation from the lacUV5 promoter.
Figure 3: Dissociation and binding of σ70 subunit in TEC16 or TEC17.
Figure 4: Mapping of RNAP contacts with DNA of transcription bubble.
Figure 5: Specific interaction of σ70 with the initially transcribed region of lacUV5 promoter.

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References

  1. Young, B.A., Gruber, T.M. & Gross, C.A. Views of transcription initiation. Cell 109, 417–420 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Lonetto, M., Gribskov, M. & Gross, C.A. The σ70 family, sequence conservation and evolutionary relationships. J. Bacteriol. 174, 3843–3849 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hawley, D.K. & McClure, W.R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids. Res. 11, 2237–2255 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Travers, A. & Burgess, R. Cyclic re-use of the RNA polymerase σ factor. Nature 222, 537–540 (1969).

    Article  CAS  PubMed  Google Scholar 

  5. Hansen, U.M. & McClure, W.R. Role of the σ subunit of Escherichia coli RNA polymerase in initiation. II. Release of σ from ternary complexes. J. Biol. Chem. 255, 9564–9570 (1980).

    CAS  PubMed  Google Scholar 

  6. Krummel, B. & Chamberlin, M.J. RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry 28, 7829–7842 (1989).

    Article  CAS  PubMed  Google Scholar 

  7. Metzger, W., Schickor, P., Meier, T., Werel, W. & Heumann, H. Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase. J. Mol. Biol. 232, 35–49 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Ring, B.Z., Yarnell, W.S. & Roberts, J.W. Function of E. coli RNA polymerase σ factor σ70 in promoter-proximal pausing. Cell 86, 485–493 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Bar-Nahum, G. & Nudler, E. Isolation and characterization of σ70-retaining transcription elongation complexes from Escherichia coli. Cell 106, 443–451 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Mukhopadhyay, J. et al. Translocation of σ70 with RNA polymerase during transcription. Fluorescence resonance energy transfer assay for movement relative to DNA. Cell 106, 453–463 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Murakami, K.S., Masuda, S. & Darst, S.A. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280–1284 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Vassylyev, D.G. et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712–719 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Shimamoto, N., Kamigochi, T. & Utiyama, H. Release of the σ subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA. J. Biol. Chem. 261, 11859–11865 (1986).

    CAS  PubMed  Google Scholar 

  14. Nickels, B.E., Roberts, C.W., Sun, H., Roberts, J.W. & Hochschild, A. The σ70 subunit of RNA polymerase is contacted by the λQ antiterminator during early elongation. Mol. Cell 10, 611–622 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Brodolin, K. & Buckle, M. Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in open transcription complexes. J. Mol. Biol. 307, 25–30 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Brodolin, K., Mustaev, A., Severinov, K. & Nikiforov, V. Identification of RNA polymerase β′ subunit segment contacting the melted region of the lacUV5 promoter. J. Biol. Chem. 275, 3661–3666 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Severinov, K. et al. Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the β and β′ subunits of Escherichia coli RNA polymerase. J. Biol. Chem. 271, 27969–27974 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Pfeffer, S.R., Stahl, S.J. & Chamberlin, M.J. Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin. J. Biol. Chem. 252, 5403–5407 (1977).

    CAS  PubMed  Google Scholar 

  19. Cech, C.L. & McClure, W.R. Characterization of ribonucleic acid polymerase-T7 promoter binary complexes. Biochemistry 19, 2440–2447 (1980).

    Article  CAS  PubMed  Google Scholar 

  20. Fisher, R. & Blumenthal, T. Analysis of RNA polymerase by trypsin cleavage. Evidence for a specific association between subunits σ and β involved in the closed to open complex transition. J. Biol. Chem. 255, 11056–11062 (1980).

    CAS  PubMed  Google Scholar 

  21. Marr, M.T. & Roberts, J.W. Function of transcription cleavage factors GreA and GreB at a regulatory pause site. Mol. Cell 6, 1275–1285 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Carpousis, A.J. & Gralla, J.D. Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. J. Mol. Biol. 183, 165–177 (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Borukhov, S., Sagitov, V. & Goldfarb, A. Transcript cleavage factors from E. coli. Cell 72, 459–466 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Gill, S.C., Weitzel, S.E. & von Hippel, P.H. Escherichia coli σ70 and NusA proteins. I. Binding interactions with core RNA polymerase in solution and within the transcription complex. J. Mol. Biol. 220, 307–324 (1991).

    Article  CAS  PubMed  Google Scholar 

  25. Sasse-Dwight, S. & Gralla, J.D. KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J. Biol. Chem. 264, 8074–8081 (1989).

    CAS  PubMed  Google Scholar 

  26. Siegele, D.A., Hu, J.C., Walter, W.A. & Gross, C.A. Altered promoter recognition by mutant forms of the σ70 subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206, 591–603 (1989).

    Article  CAS  PubMed  Google Scholar 

  27. Waldburger, C., Gardella, T., Wong, R. & Susskind, M.M. Changes in conserved region 2 of Escherichia coli σ70 affecting promoter recognition. J. Mol. Biol. 215, 267–276 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Juang, Y.-L. & Helmann, J.D. A promoter melting region in the primary σ factor of Bacillus subtilis. Identification of functionally important aromatic amino acids. J. Mol. Biol. 235, 1470–1488 (1994).

    Article  CAS  PubMed  Google Scholar 

  29. Roberts, C.W. & Roberts, J.W. Base-specific recognition of the nontemplate strand of promoter DNA by E. coli RNA polymerase. Cell 86, 495–501 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Gilbert, W. & Maxam, A. The nucleotide sequence of the lac operator. Proc. Natl. Acad. Sci. USA 70, 3581–3584 (1973).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Young, B.A. et al. A coiled-coil from the RNA polymerase β′ subunit allosterically induces selective nontemplate strand binding by σ70. Cell 105, 935–944 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Nickels, B.E., Mukhopadhyay, J., Garrity, S.J., Elbright, R.H. & Hochschild, A. The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter. Nat. Struct. Mol. Biol. advance online publication, 2 May 2004 (doi:10.1038/nsmb757)

  33. Karls et al. Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters. Nucleic Acids Res. 17, 3927–3949 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ozoline, O.N., Deev, A.A., Arkhipova, M.V., Chasov, V.V. & Travers, A. Proximal transcribed regions of bacterial promoters have a non-random distribution of A/T tracts. Nucleic Acids Res. 27, 4768–4774 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Marr, M.T., Datwyler, S.A., Meares, C.F. & Roberts, J.W. Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins. Proc. Natl. Acad. Sci. USA 98, 8972–8978 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mooney, R.A. & Landick, R. Tethering σ70 to RNA polymerase reveals high in vivo activity of σ factors and σ70-dependent pausing at promoter-distal locations. Genes Dev. 17, 2839–2851 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nugent, M.A. Heparin sequencing brings structure to the function of complex oligosaccharides. Proc. Natl. Acad. Sci. USA 97, 10301–10303 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Borukhov, S. et al. Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the β subunit of Escherichia coli RNA polymerase. J. Biol. Chem. 266, 23921–23926 (1991).

    CAS  PubMed  Google Scholar 

  39. Mooney, R.A. & Landick, R. RNA polymerase unveiled. Cell 98, 687–690 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Yarnell, W.S. & Roberts, J.W. The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase. Cell 69, 1181–1189 (1992).

    Article  CAS  PubMed  Google Scholar 

  41. Wassarman, K.M. & Storz, G. 6S RNA regulates E. coli RNA polymerase activity. Cell 101, 613–623 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Gribskov, M. & Burgess, R.R. Overexpression and purification of the σ subunit of Escherichia coli RNA polymerase. Gene 26, 109–118 (1983).

    Article  CAS  PubMed  Google Scholar 

  43. Keilty, S. & Rosenberg, M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J. Biol. Chem. 262, 6389–6395 (1987).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Russian Foundation for Basic Research grant 02-04-48525. K.B. was supported by a grant from INTAS (YSF220) and a German Academic Exchange Service (DAAD) travel grant while in the laboratory of H.H. We are grateful to A. Hochschild, K. Severinov, D. Fisher and D. Maiorano for critical reading of the manuscript.

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  1. Konstantin Brodolin

    Present address: Institut de Génétique Humaine, 141 rue de la Cardonille, 34396, Montpellier, France

Authors and Affiliations

  1. Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Square 2, Moscow, 123182, Russia

    Konstantin Brodolin & Nikolay Zenkin

  2. Public Health Research Institute, 255 Warren Street, Newark, New Jersey, USA

    Arkady Mustaev

  3. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow, Russia

    Daria Mamaeva

  4. Max-Planck-Institut für Biochemie, Martinsried bei Muenchen, Am Klopferspitz 18A, D 82152, Germany

    Hermann Heumann

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Correspondence to Konstantin Brodolin.

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Brodolin, K., Zenkin, N., Mustaev, A. et al. The σ70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription. Nat Struct Mol Biol 11, 551–557 (2004). https://doi.org/10.1038/nsmb768

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