Skip to main content
SpringerLink
Log in

The σ24 Subunit of Escherichia coli RNA Polymerase Can Induce Transcriptional Pausing in vitro

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the–10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding–10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the–10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

nt:

nucleotides

RNAP:

RNA polymerase

TEC:

transcription elongation complex

References

  1. Feklistov, A., Sharon, B. D., Darst, S. A., and Gross, C. A. (2014) Bacterial sigma factors: a historical, structural, and genomic perspective, Annu. Rev. Microbiol., 68, 357–376.

    Article  CAS  PubMed  Google Scholar 

  2. Gruber, T. M., and Gross, C. A. (2003) Multiple sigma sub–units and the partitioning of bacterial transcription space, Annu. Rev. Microbiol., 57, 441–466.

    Article  CAS  PubMed  Google Scholar 

  3. Paget, M. S. (2015) Bacterial sigma factors and anti–sigma factors: structure, function and distribution, Biomolecules, 5, 1245–1265.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang, N., Darbari, V. C., Glyde, R., Zhang, X., and Buck, M. (2016) The bacterial enhancer–dependent RNA poly–merase, Biochem. J., 473, 3741–3753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lonetto, M., Gribskov, M., and Gross, C. A. (1992) The sigma 70 family: sequence conservation and evolutionary relationships, J. Bacteriol., 174, 3843–3849.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Iyer, L. M., and Aravind, L. (2012) Insights from the archi–tecture of the bacterial transcription apparatus, J. Struct. Biol., 179, 299–319.

    Article  CAS  PubMed  Google Scholar 

  7. Maciag, A., Peano, C., Pietrelli, A., Egli, T., De Bellis, G., and Landini, P. (2011) In vitro transcription profiling of the sigmaS subunit of bacterial RNA polymerase: re–definition of the sigmaS regulon and identification of sigmaS–specific pro–moter sequence elements, Nucleic Acids Res., 39, 5338–5355.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, B., Zuo, Y., and Steitz, T. A. (2016) Structures of E. coli sigmaS–transcription initiation complexes provide new insights into polymerase mechanism, Proc. Natl. Acad. Sci. USA, 113, 4051–4056.

    Article  CAS  PubMed  Google Scholar 

  9. White–Ziegler, C. A., Um, S., Perez, N. M., Berns, A. L., Malhowski, A. J., and Young, S. (2008) Low temperature (23°C) increases expression of biofilm–, cold–shock–and RpoS–dependent genes in Escherichia coli K–12, Microbiology, 154, 148–166.

    Article  CAS  PubMed  Google Scholar 

  10. Battesti, A., Majdalani, N., and Gottesman, S. (2011) The RpoS–mediated general stress response in Escherichia coli, Annu. Rev. Microbiol., 65, 189–213.

    Article  CAS  PubMed  Google Scholar 

  11. Zhao, K., Liu, M., and Burgess, R. R. (2007) Adaptation in bacterial flagellar and motility systems: from regulon mem–bers to “foraging”–like behavior in E. coli, Nucleic Acids Res., 35, 4441–4452.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nonaka, G., Blankschien, M., Herman, C., Gross, C. A., and Rhodius, V. A. (2006) Regulon and promoter analysis of the E. coli heat–shock factor, sigma32, reveals a multifac–eted cellular response to heat stress, Genes Dev., 20, 1776–1789.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Neidhardt, F. C., VanBogelen, R. A., and Lau, E. T. (1983) Molecular cloning and expression of a gene that controls the high–temperature regulon of Escherichia coli, J. Bacteriol., 153, 597–603.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Grossman, A. D., Erickson, J. W., and Gross, C. A. (1984) The htpR gene product of E. coli is a sigma factor for heat–shock promoters, Cell, 38, 383–390.

    Article  CAS  PubMed  Google Scholar 

  15. Komeda, Y. (1986) Transcriptional control of flagellar genes in Escherichia coli K–12, J. Bacteriol., 168, 1315–1318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Komeda, Y., Kutsukake, K., and Iino, T. (1980) Definition of additional flagellar genes in Escherichia coli K12, Genetics, 94, 277–290.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Arnosti, D. N., and Chamberlin, M. J. (1989) Secondary sigma factor controls transcription of flagellar and chemo–taxis genes in Escherichia coli, Proc. Natl. Acad. Sci. USA, 86, 830–834.

    Article  CAS  PubMed  Google Scholar 

  18. Barrios, A. F., Zuo, R., Ren, D., and Wood, T. K. (2006) Hha, YbaJ, and OmpA regulate Escherichia coli K12 biofilm formation and conjugation plasmids abolish motil–ity, Biotechnol. Bioeng., 93, 188–200.

    CAS  PubMed  Google Scholar 

  19. Lipinska, B., Sharma, S., and Georgopoulos, C. (1988) Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32–independent mechanism of heat–inducible transcription, Nucleic Acids Res., 16, 10053–10067.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, Q. P., and Kaguni, J. M. (1989) A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli, J. Bacteriol., 171, 4248–4253.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Rouviere, P. E., De Las Penas, A., Mecsas, J., Lu, C. Z., Rudd, K. E., and Gross, C. A. (1995) rpoE, the gene encoding the second heat–shock sigma factor, sigma E, in Escherichia coli, EMBO J., 14, 1032–1042.

    CAS  Google Scholar 

  22. Egler, M., Grosse, C., Grass, G., and Nies, D. H. (2005) Role of the extracytoplasmic function protein family sigma factor RpoE in metal resistance of Escherichia coli, J. Bacteriol., 187, 2297–2307.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Angerer, A., Enz, S., Ochs, M., and Braun, V. (1995) Transcriptional regulation of ferric citrate transport in Escherichia coli K–12. Fecl belongs to a new subfamily of sigma 70–type factors that respond to extracytoplasmic stimuli, Mol. Microbiol., 18, 163–174.

    Article  CAS  PubMed  Google Scholar 

  24. Bar–Nahum, G., and Nudler, E. (2001) Isolation and char–acterization of sigma(70)–retaining transcription elonga–tion complexes from Escherichia coli, Cell, 106, 443–451.

    Article  PubMed  Google Scholar 

  25. Kapanidis, A. N., Margeat, E., Laurence, T. A., Doose, S., Ho, S. O., Mukhopadhyay, J., Kortkhonjia, E., Mekler, V., Ebright, R. H., and Weiss, S. (2005) Retention of tran–scription initiation factor sigma70 in transcription elonga–tion: single–molecule analysis, Mol. Cell, 20, 347–356.

    Article  CAS  PubMed  Google Scholar 

  26. Mukhopadhyay, J., Kapanidis, A. N., Mekler, V., Kortkhonjia, E., Ebright, Y. W., and Ebright, R. H. (2001) Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA, Cell, 106, 453–463.

    Article  CAS  PubMed  Google Scholar 

  27. Mooney, R. A., Davis, S. E., Peters, J. M., Rowland, J. L., Ansari, A. Z., and Landick, R. (2009) Regulator trafficking on bacterial transcription units in vivo, Mol. Cell, 33, 97–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Raffaelle, M., Kanin, E. I., Vogt, J., Burgess, R. R., and Ansari, A. Z. (2005) Holoenzyme switching and stochastic release of sigma factors from RNA polymerase in vivo, Mol. Cell, 20, 357–366.

    Article  CAS  PubMed  Google Scholar 

  29. Harden, T. T., Wells, C. D., Friedman, L. J., Landick, R., Hochschild, A., Kondev, J., and Gelles, J. (2016) Bacterial RNA polymerase can retain sigma70 throughout transcrip–tion, Proc. Natl. Acad. Sci. USA, 113, 602–607.

    Article  CAS  PubMed  Google Scholar 

  30. Brodolin, K., Zenkin, N., Mustaev, A., Mamaeva, D., and Heumann, H. (2004) The sigma 70 subunit of RNA poly–merase induces lacUV5 promoter–proximal pausing of transcription, Nat. Struct. Mol. Biol., 11, 551–557.

    Article  CAS  PubMed  Google Scholar 

  31. Goldman, S. R., Nair, N. U., Wells, C. D., Nickels, B. E., and Hochschild, A. (2015) The primary sigma factor in Escherichia coli can access the transcription elongation complex from solution in vivo, eLife, 4, e10514.

    Google Scholar 

  32. Mooney, R. A., Darst, S. A., and Landick, R. (2005) Sigma and RNA polymerase: an on–again, off–again relationship? Mol. Cell, 20, 335–345.

    Article  CAS  PubMed  Google Scholar 

  33. Zhilina, E., Esyunina, D., Brodolin, K., and Kulbachinskiy, A. (2012) Structural transitions in the tran–scription elongation complexes of bacterial RNA poly–merase during sigma–dependent pausing, Nucleic Acids Res., 40, 3078–3091.

    Article  CAS  PubMed  Google Scholar 

  34. Petushkov, I., Esyunina, D., and Kulbachinskiy, A. (2017) Sigma38–dependent promoter–proximal pausing by bacter–ial RNA polymerase, Nucleic Acids Res., 45, 3006–3016.

    Article  CAS  PubMed  Google Scholar 

  35. Perdue, S. A., and Roberts, J. W. (2011) Sigma(70)–dependent transcription pausing in Escherichia coli, J. Mol. Biol., 412, 782–792.

    Article  CAS  PubMed  Google Scholar 

  36. Ring, B. Z., Yarnell, W. S., and Roberts, J. W. (1996) Function of E. coli RNA polymerase sigma factor sigma 70 in promoter–proximal pausing, Cell, 86, 485–493.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  38. Nickels, B. E., Mukhopadhyay, J., Garrity, S. J., Ebright, R. H., and Hochschild, A. (2004) The sigma 70 subunit of RNA polymerase mediates a promoter–proximal pause at the lac promoter, Nat. Struct. Mol. Biol., 11, 544–550.

    Article  CAS  PubMed  Google Scholar 

  39. Zenkin, N., Kulbachinskiy, A., Yuzenkova, Y., Mustaev, A., Bass, I., Severinov, K., and Brodolin, K. (2007) Region 1.2 of the RNA polymerase sigma subunit controls recognition of the–10 promoter element, EMBO J., 26, 955–964.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Devi, P. G., Campbell, E. A., Darst, S. A., and Nickels, B. E. (2010) Utilization of variably spaced promoter–like ele–ments by the bacterial RNA polymerase holoenzyme dur–ing early elongation, Mol. Microbiol., 75, 607–622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Perdue, S. A., and Roberts, J. W. (2010) A backtrack–inducing sequence is an essential component of Escherichia coli sigma(70)–dependent promoter–proximal pausing, Mol. Microbiol., 78, 636–650.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Strobel, E. J., and Roberts, J. W. (2014) Regulation of pro–moter–proximal transcription elongation: enhanced DNA scrunching drives lambdaQ antiterminator–dependent escape from a sigma70–dependent pause, Nucleic Acids Res., 42, 5097–5108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Deighan, P., Pukhrambam, C., Nickels, B. E., and Hochschild, A. (2011) Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex, Genes Dev., 25, 77–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Petushkov, I., Esyunina, D., and Kulbachinskiy, A. (2017) Possible roles of sigma–dependent RNA polymerase paus–ing in transcription regulation, RNA Biol., 14, 1678–1682.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Svetlov, V., and Artsimovitch, I. (2015) Purification of bac–terial RNA polymerase: tools and protocols, Methods Mol. Biol., 1276, 13–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pupov, D., Kuzin, I., Bass, I., and Kulbachinskiy, A. (2014) Distinct functions of the RNA polymerase sigma subunit region 3.2 in RNA priming and promoter escape, Nucleic Acids Res., 42, 4494–4504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Anthony, L. C., Foley, K. M., Thompson, N. E., and Burgess, R. R. (2003) Expression, purification of, and monoclonal antibodies to sigma factors from Escherichia coli, Methods Enzymol., 370, 181–192.

    Article  CAS  PubMed  Google Scholar 

  48. Laptenko, O., and Borukhov, S. (2003) Biochemical assays of Gre factors of Thermus thermophilus, Methods Enzymol., 371, 219–232.

    Article  CAS  PubMed  Google Scholar 

  49. Rhodius, V. A., Suh, W. C., Nonaka, G., West, J., and Gross, C. A. (2006) Conserved and variable functions of the sigmaE stress response in related genomes, PLoS Biol., 4, e2.

    Google Scholar 

  50. Strobel, E. J., and Roberts, J. W. (2015) Two transcription pause elements underlie a sigma70–dependent pause cycle, Proc. Natl. Acad. Sci. USA, 112, E4374–4380.

    Google Scholar 

  51. Campagne, S., Marsh, M. E., Capitani, G., Vorholt, J. A., and Allain, F. H. (2014) Structural basis for–10 promoter element melting by environmentally induced sigma factors, Nat. Struct. Mol. Biol., 21, 269–276.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  54. Laptenko, O., Lee, J., Lomakin, I., and Borukhov, S. (2003) Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase, EMBO J., 22, 6322–6334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Grigorova, I. L., Phleger, N. J., Mutalik, V. K., and Gross, C. A. (2006) Insights into transcriptional regulation and sigma competition from an equilibrium model of RNA polymerase binding to DNA, Proc. Natl. Acad. Sci. USA, 103, 5332–5337.

    Article  CAS  PubMed  Google Scholar 

  56. Jishage, M., Iwata, A., Ueda, S., and Ishihama, A. (1996) Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions, J. Bacteriol., 178, 5447–5451.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhilina, E., Miropolskaya, N., Bass, I., Brodolin, K., and Kulbachinskiy, A. (2011) Characteristics of sigma–depend–ent pausing in RNA polymerases from E. coli and T. aquati–cus, Biochemistry (Moscow), 76, 1348–1358.

    Article  CAS  Google Scholar 

  58. Hatoum, A., and Roberts, J. (2008) Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation, Mol. Microbiol., 68, 17–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

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

    A. B. Shikalov, D. M. Esyunina, D. V. Pupov, A. V. Kulbachinskiy & I. V. Petushkov

Authors
  1. A. B. Shikalov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. M. Esyunina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. V. Pupov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. V. Kulbachinskiy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. I. V. Petushkov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to I. V. Petushkov.

Additional information

Published in Russian in Biokhimiya, 2019, Vol. 84, No. 4, pp. 571–579.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18–301, February 11, 2019.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shikalov, A.B., Esyunina, D.M., Pupov, D.V. et al. The σ24 Subunit of Escherichia coli RNA Polymerase Can Induce Transcriptional Pausing in vitro. Biochemistry Moscow 84, 426–434 (2019). https://doi.org/10.1134/S0006297919040102

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297919040102

Keywords

Search

Navigation