Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Role of the RNA polymerase trigger loop in catalysis and pausing

Nature Structural & Molecular Biology volume 17, pages 99–104 (2010)Cite this article

Abstract

The trigger loop (TL) is a polymorphous component of RNA polymerase (RNAP) that makes direct substrate contacts and promotes nucleotide addition when folded into an α-helical hairpin (trigger helices, TH). However, the roles of the TL/TH in transcript cleavage, catalysis, substrate selectivity and pausing remain ill defined. Based on in vitro assays of Escherichia coli RNAP bearing specific TL/TH alterations, we report that neither intrinsic nor regulator-assisted transcript cleavage of backtracked RNA requires formation of the TH. We find that the principal contribution of TH formation to rapid nucleotidyl transfer is steric alignment of the reactants rather than acid-base catalysis, and that the TL/TH cannot be the sole contributor to substrate selectivity. The similar effects of TL/TH substitutions on pausing and nucleotide addition provide additional support for the view that TH formation is rate-limiting for escape from nonbacktracked pauses.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nucleotidyl transfer and RNA hydrolysis reactions catalyzed by multisubunit RNAPs.
Figure 2: The TL is dispensable for intrinsic RNA hydrolysis on a locked scaffold.
Figure 3: GreB requires SI3 but not TL folding to stimulate transcript hydrolysis.
Figure 4: Significant discrimination against 2′ dNTPs is retained in ΔTL RNAP.
Figure 5: TL folding is similarly rate-limiting for rapid nucleotide addition, his pause escape and pyrophosphorolysis.
Figure 6: TL dynamics in nucleotidyl transfer, transcriptional pausing and transcript cleavage.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Iyer, L.M., Koonin, E.V. & Aravind, L. Evolution of bacterial RNA polymerase: implications for large-scale bacterial phylogeny, domain accretion, and horizontal gene transfer. Gene 335, 73–88 (2004).

    Article  CAS  Google Scholar 

  2. Kireeva, M.L. et al. Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation. Mol. Cell 30, 557–566 (2008).

    Article  CAS  Google Scholar 

  3. Landick, R. Active-site dynamics in RNA polymerases. Cell 116, 351–353 (2004).

    Article  CAS  Google Scholar 

  4. Rhodes, G. & Chamberlin, M.J. Ribonucleic acid chain elongation by Escherichia coli ribonucleic acid polymerase: isolation of ternary complexes and the kinetics of elongation. J. Biol. Chem. 249, 6675–6683 (1974).

    CAS  PubMed  Google Scholar 

  5. Richardson, J.P. Loading Rho to terminate transcription. Cell 114, 157–159 (2003).

    Article  CAS  Google Scholar 

  6. Vassylyev, D. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 157–162 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Wang, D., Bushnell, D.A., Westover, K.D., Kaplan, C.D. & Kornberg, R.D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006).

    Article  CAS  Google Scholar 

  9. Yarnell, W.S. & Roberts, J.W. Mechanism of instrinsic termination and antitermination. Science 284, 611–615 (1999).

    Article  CAS  Google Scholar 

  10. Zenkin, N., Yuzenkova, Y. & Severinov, K. Transcript-assisted transcriptional proofreading. Science 313, 518–520 (2006).

    Article  CAS  Google Scholar 

  11. Sosunov, V. et al. Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase. EMBO J. 22, 2234–2244 (2003).

    Article  CAS  Google Scholar 

  12. Kettenberger, H., Armache, K.J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004).

    Article  CAS  Google Scholar 

  13. Opalka, N. et al. Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase. Cell 114, 335–345 (2003).

    Article  CAS  Google Scholar 

  14. Orlova, M., Newlands, J., Das, A., Goldfarb, A. & Borukhov, S. Intrinsic transcript cleavage activity of RNA polymerase. Proc. Natl. Acad. Sci. USA 92, 4596–4600 (1995).

    Article  CAS  Google Scholar 

  15. Sosunova, E. et al. Donation of catalytic residues to RNA polymerase active center by transcription factor Gre. Proc. Natl. Acad. Sci. USA 100, 15469–15474 (2003).

    Article  CAS  Google Scholar 

  16. Wang, D. et al. Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution. Science 324, 1203–1206 (2009).

    Article  CAS  Google Scholar 

  17. Steitz, T.A. A mechanism for all polymerases. Nature 391, 231–232 (1998).

    Article  CAS  Google Scholar 

  18. Mustaev, A. et al. Modular organization of the catalytic center of RNA polymerase. Proc. Natl. Acad. Sci. USA 94, 6641–6645 (1997).

    Article  CAS  Google Scholar 

  19. Castro, C. et al. Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. Proc. Natl. Acad. Sci. USA 104, 4267–4272 (2007).

    Article  CAS  Google Scholar 

  20. Castro, C. et al. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. 16, 212–218 (2009).

    Article  CAS  Google Scholar 

  21. Tan, L., Wiesler, S., Trzaska, D., Carney, H.C. & Weinzierl, R.O. Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J. Biol. 7, 40–54 (2008).

    Article  Google Scholar 

  22. Toulokhonov, I., Zhang, J., Palangat, M. & Landick, R. A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing. Mol. Cell 27, 406–419 (2007).

    Article  CAS  Google Scholar 

  23. Artsimovitch, I., Svetlov, V., Murakami, K. & Landick, R. Co-overexpression of E. coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions. J. Biol. Chem. 278, 12344–12355 (2003).

    Article  CAS  Google Scholar 

  24. Chlenov, M. et al. Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase β′ subunit. J. Mol. Biol. 353, 138–154 (2005).

    Article  CAS  Google Scholar 

  25. Brueckner, F. & Cramer, P. Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation. Nat. Struct. Mol. Biol. 15, 811–818 (2008).

    Article  CAS  Google Scholar 

  26. Kaplan, C.D., Larsson, K.M. & Kornberg, R.D. The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. Mol. Cell 30, 547–556 (2008).

    Article  CAS  Google Scholar 

  27. Temiakov, D. et al. Structural basis of transcription inhibition by antibiotic streptolydigin. Mol. Cell 19, 655–666 (2005).

    Article  CAS  Google Scholar 

  28. Tuske, S. et al. Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation. Cell 122, 541–552 (2005).

    Article  CAS  Google Scholar 

  29. Herbert, K.M. et al. Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125, 1083–1094 (2006).

    Article  CAS  Google Scholar 

  30. Neuman, K.C., Abbondanzieri, E.A., Landick, R., Gelles, J. & Block, S.M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115, 437–447 (2003).

    Article  CAS  Google Scholar 

  31. Svetlov, V., Vassylyev, D.G. & Artsimovitch, I. Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase. J. Biol. Chem. 279, 38087–38090 (2004).

    Article  CAS  Google Scholar 

  32. Malagon, F. et al. Mutations in the Saccharomyces cerevisiae RPB1 gene conferring hypersensitivity to 6-azauracil. Genetics 172, 2201–2209 (2006).

    Article  CAS  Google Scholar 

  33. Bar-Nahum, G. et al. A ratchet mechanism of transcription elongation and its control. Cell 120, 183–193 (2005).

    Article  CAS  Google Scholar 

  34. Kireeva, M.L. & Kashlev, M. Mechanism of sequence-specific pausing of bacterial RNA polymerase. Proc. Natl. Acad. Sci. USA 106, 8900–8905 (2009).

    Article  CAS  Google Scholar 

  35. Landick, R. Transcriptional pausing without backtracking. Proc. Natl. Acad. Sci. USA 106, 8797–8798 (2009).

    Article  CAS  Google Scholar 

  36. Kraynov, V.S., Showalter, A.K., Liu, J., Zhong, X. & Tsai, M.D. DNA polymerase β: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity. Biochemistry 39, 16008–16015 (2000).

    Article  CAS  Google Scholar 

  37. Toulokhonov, I. & Landick, R. The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. Mol. Cell 12, 1125–1136 (2003).

    Article  CAS  Google Scholar 

  38. Chan, C.L., Wang, D. & Landick, R. Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3′ end spacing distinguishes pause and termination pathways. J. Mol. Biol. 268, 54–68 (1997).

    Article  CAS  Google Scholar 

  39. Sydow, J.F. et al. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell 34, 710–721 (2009).

    Article  CAS  Google Scholar 

  40. Vassylyev, D.G., Vassylyeva, M.N., Perederina, A., Tahirov, T.H. & Artsimovitch, I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007).

    Article  CAS  Google Scholar 

  41. Wang, D. et al. Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex. Cell 81, 341–350 (1995).

    Article  CAS  Google Scholar 

  42. Kyzer, S., Ha, K.S., Landick, R. & Palangat, M. Direct versus limited-step reconstitution reveals key features of an RNA hairpin-stabilized paused transcription complex. J. Biol. Chem. 282, 19020–19028 (2007).

    Article  CAS  Google Scholar 

  43. Sidorenkov, I., Komissarova, N. & Kashlev, M. Crucial role of the RNA:DNA hybrid in the processivity of transcription. Mol. Cell 2, 55–64 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Landick laboratory for many suggestions during the course of this work. This work was supported by US National Institutes of Health grant GM38660 to R.L.

Author information

Author notes

  1. Jinwei Zhang

    Present address: Present address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (J.Z.).,

Authors and Affiliations

  1. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Jinwei Zhang

  2. Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA

    Murali Palangat & Robert Landick

  3. Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA

    Robert Landick

Authors
  1. Jinwei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Murali Palangat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert Landick
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.Z. constructed the mutant RNAPs, purified the proteins, performed elongation, pausing, cleavage and misincorporation assays and contributed to interpretation of results and preparation of the manuscript. P.M. performed the rapid-mixer kinetic experiments and contributed to interpretation of results and preparation of the manuscript. R.L. conceived and coordinated the project and wrote the paper.

Corresponding author

Correspondence to Robert Landick.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 402 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, J., Palangat, M. & Landick, R. Role of the RNA polymerase trigger loop in catalysis and pausing. Nat Struct Mol Biol 17, 99–104 (2010). https://doi.org/10.1038/nsmb.1732

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1732

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing