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The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter

Nature Structural & Molecular Biology volume 11, pages 544–550 (2004)Cite this article

Abstract

The σ70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, σ70 has a critical regulatory role during transcription elongation at the bacteriophage λ late promoter, λPR′. At this promoter, σ70 mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter −10 element. Here we provide evidence that σ70 also mediates a pause in early elongation at the lac promoter (plac). Like that at λPR′, the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter −10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between σ70 and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal σ70-dependent pauses may play a role in the regulation of many bacterial promoters.

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Figure 1: In vitro transcription assays with λPR′ and plac templates.
Figure 2: Sequence element in the initially transcribed region of plac mediates a σ70-dependent pause in vitro.
Figure 3: The placUV5 paused elongation complex is sensitive to GreB and GreA.
Figure 4: Sequence element in the initially transcribed region of plac mediates a GreA-sensitive σ-dependent pause in vivo.
Figure 5: Sequence element in the initially transcribed region of plac stabilizes interactions between σ70 and the remainder of the transcription elongation complex.

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References

  1. Gross, C.A. et al. The functional and regulatory roles of σ factors in transcription. Cold Spring Harbor Symp. Quant. Biol. 63, 141–155 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Straney, D. & Crothers, D. A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J. Mol. Biol. 193, 267–278 (1987).

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

  5. 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  Google Scholar 

  6. Daube, S. & von Hippel, P. Interactions of Escherichia coli σ70 within the transcription elongation complex. Proc. Natl. Acad. Sci. USA 96, 8390–8395 (1999)..

    Article  CAS  Google Scholar 

  7. Hansen, U.M. & McClure, W.R. Role of the σ70 subunit of Escherichia coli RNA polymerase in initiation. II. Release of σ70 from ternary complexes. J. Biol. Chem. 255, 9564–9570 (1980).

    CAS  PubMed  Google Scholar 

  8. Mekler, V. et al. Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599–614 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. 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 

  13. Osumi-Davis, P.A., Woody, A.Y. & Woody, R.W. Transcription initiation by Escherichia coli RNA polymerase at the gene II promoter of M13 phage: stability of ternary complex, direct photocrosslinking to nascent RNA, and retention of σ subunit. Biochim. Biophys. Acta. 910, 130–141 (1987).

    Article  CAS  Google Scholar 

  14. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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  Google Scholar 

  17. Roberts, J.W. et al. Antitermination by bacteriophage λ Q protein. Cold Spring Harbor Symp. Quant. Biol. 63, 319–325 (1998).

    Article  CAS  Google Scholar 

  18. Burr, T., Mitchell, J., Kolb, A., Minchin, S. & Busby, S. DNA sequence elements located immediately upstream of the −10 hexamer in Escherichia coli promoters: a systematic study. Nucleic Acids Res. 28, 1864–1870 (2000).

    Article  CAS  Google Scholar 

  19. Arditti, R.R., Scaife, J.G. & Beckwith, J.R. The nature of mutants in the lac promoter region. J. Mol. Biol. 38, 421–426 (1968).

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Grayhack, E.J., Yang, X.J., Lau, L.F. & Roberts, J.W. Phage λ gene-Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site. Cell 42, 259–269 (1985).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Ko, D.C., Marr, M.T., Guo, T.S. & Roberts, J.W. A surface of Escherichia coli σ70 required for promoter function and antitermination by phage λ Q protein. Genes Dev. 12, 3276–3285 (1998).

    Article  CAS  Google Scholar 

  24. Maizels, N.M. The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proc. Natl. Acad. Sci. USA 70, 3585–3589 (1973).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Fish, R.N. & Kane, C.M. Promoting elongation with transcript cleavage stimulatory factors. Biochim. Biophys. Acta. 1577, 287–307 (2002).

    Article  CAS  Google Scholar 

  27. Mukhopadhyay, J. et al. Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex structure and function. Methods Enzymol. 371, 144–159 (2003).

    Article  CAS  Google Scholar 

  28. Brodolin, K., Zenkin, N., Mustaev, A., Mamaeva, D. & Heumann, H. σ70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription. Nat. Struct. Mol. Biol. 11, advance online publication, 2 May 2004 (doi:10.1038/nsmb768) (2004).

  29. Karls, R. et al. Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters. Nucleic Acids Res. 17, 3927–3939 (1989).

    Article  CAS  Google Scholar 

  30. Ring, B.Z. & Roberts, J.W. Function of a nontranscribed DNA strand site in transcription elongation. Cell 78, 317–324 (1994).

    Article  CAS  Google Scholar 

  31. Salgado, H. et al. RegulonDB (version 4.0): transcriptional regulation, operon organization and growth conditions in Escherichia coli K-12. Nucleic Acids Res. 32, D303–D306 (2004).

    Article  CAS  Google Scholar 

  32. Epshtein, V. & Nudler, E. Cooperation between RNA polymerase molecules in transcription elongation. Science 300, 801–805 (2003).

    Article  CAS  Google Scholar 

  33. Epshtein, V., Toulme, F., Rahmouni, A.R., Borukhov, S. & Nudler, E. Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727 (2003).

    Article  CAS  Google Scholar 

  34. 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  Google Scholar 

  35. Whipple, F.W. Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli. Nucleic Acids Res. 26, 3700–3706 (1998).

    Article  CAS  Google Scholar 

  36. Miller, J.H. In Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, Plainview, New York, 1972).

    Google Scholar 

  37. Panaghie, G., Aiyar, S.E., Bobb, K.L., Hayward, R.S. & de Haseth, P.L. Aromatic amino acids in region 2.3 of Escherichia coli σ70 participate collectively in the formation of an RNA polymerase-promoter open complex. J. Mol. Biol. 299, 1217–1230 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Roberts, K. Brodolin, A. Kapanidis and S. Weiss for discussion, S. Borukhov for purified GreA and GreB, A. Huerta and J. Collado-Vides for providing promoter sequences, S. Dove for comments on the manuscript, and R. Hellmiss for assistance with figure preparation. We also thank A. Hatoum and J. Roberts for sharing unpublished results. This work was supported by US National Institutes of Health grant GM44025 to A.H. and GM41376 to R.H.E. and by a Howard Hughes Medical Institute investigatorship to R.H.E.

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  1. Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Bryce E Nickels, Sean J Garrity & Ann Hochschild

  2. Department of Chemistry, Howard Hughes Medical Institute, Waksman Institute, Rutgers University, Piscataway, 08854, New Jersey, USA

    Jayanta Mukhopadhyay & Richard H Ebright

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Correspondence to Ann Hochschild.

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Nickels, B., Mukhopadhyay, J., Garrity, S. et al. The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter. Nat Struct Mol Biol 11, 544–550 (2004). https://doi.org/10.1038/nsmb757

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