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:

Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation

Nature Structural & Molecular Biology volume 15, pages 811–818 (2008)Cite this article

Abstract

To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor α-amanitin locked the elongation complex in a new state, which was refined at 3.4-Å resolution and identified as a possible translocation intermediate. The DNA base entering the active site occupies a 'pretemplating' position above the central bridge helix, which is shifted and occludes the templating position. A leucine residue in the trigger loop forms a wedge at the shifted bridge helix, but moves by 13 Å to close the active site during nucleotide incorporation. Our results support a Brownian ratchet mechanism that involves swinging of the trigger loop between open, wedged and closed positions, and suggest that α-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Structure of the α-amanitin–inhibited Pol II EC.
Figure 2: Contacts between α-amanitin and Pol II.
Figure 3: The α-amanitin–inhibited EC structure is apparently a translocation intermediate.
Figure 4: Comparisons with bacterial RNA polymerase and possible EC states.
Figure 5: Model of the nucleotide addition cycle (NAC).

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution. Science 292, 1876–1882 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. 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  PubMed  Google Scholar 

  3. Westover, K.D., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119, 481–489 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. 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  PubMed  PubMed Central  Google Scholar 

  5. Cramer, P., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: RNA polymerase II at 2.8 Å resolution. Science 292, 1863–1876 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. 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 

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

    Article  CAS  PubMed  Google Scholar 

  8. Epshtein, V. et al. Swing-gate model of nucleotide entry into the RNA polymerase active center. Mol. Cell 10, 623–634 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. Artsimovitch, I. et al. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins. Cell 122, 351–363 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Bushnell, D.A., Cramer, P. & Kornberg, R.D. Structural basis of transcription: α-amanitin-RNA polymerase II cocrystal at 2.8 Å resolution. Proc. Natl. Acad. Sci. USA 99, 1218–1222 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gong, X.Q., Nedialkov, Y.A. & Burton, Z.F. α-amanitin blocks translocation by human RNA polymerase II. J. Biol. Chem. 279, 27422–27427 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Vassylyev, D.G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Cramer, P. Gene transcription: extending the message. Nature 448, 142–143 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Kashkina, E. et al. Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site. J. Biol. Chem. 282, 21578–21582 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. 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  PubMed  Google Scholar 

  18. Naji, S., Bertero, M.G., Spitalny, P., Cramer, P. & Thomm, M. Structure function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement. Nucleic Acids Res. 36, 676–687 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Campbell, E.A. et al. Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J. 24, 674–682 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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  PubMed  Google Scholar 

  21. Sousa, R. Machinations of a Maxwellian demon. Cell 120, 155–156 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Damsma, G.E., Alt, A., Brueckner, F., Carell, T. & Cramer, P. Mechanism of transcriptional stalling at cisplatin-damaged DNA. Nat. Struct. Mol. Biol. 14, 1127–1133 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Temiakov, D. et al. Structural basis for substrate selection by T7 RNA polymerase. Cell 116, 381–391 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Yin, Y.W. & Steitz, T.A. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 116, 393–404 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Cramer, P. Common structural features of nucleic acid polymerases. Bioessays 24, 724–729 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Abbondanzieri, E.A., Greenleaf, W.J., Shaevitz, J.W., Landick, R. & Block, S.M. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Galburt, E.A. et al. Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446, 820–823 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Chafin, D.R., Guo, H. & Price, D.H. Actions of α-amanitin during pyrophosphoryolysis and elongation by RNA polymerase II. J. Biol. Chem. 270, 19114–19119 (1995).

    Article  CAS  PubMed  Google Scholar 

  29. Rudd, M.D. & Luse, D.S. Amanitin greatly reduces the rate of transcription by RNA polymerase II ternary complexes but fails to inhibit some transcript cleavage modes. J. Biol. Chem. 271, 21549–21558 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Wienland, T. & Faulstich, H. Fifty years of amanitin. Experientia 47, 1186–1193 (1991).

    Article  Google Scholar 

  31. Zanotti, G., Petersen, G. & Wieland, T. Structure-toxicity relationships in the amatoxin series. Int. J. Pept. Protein Res. 40, 551–558 (1992).

    Article  CAS  PubMed  Google Scholar 

  32. Armache, K.-J., Kettenberger, H. & Cramer, P. Architecture of the initiation-competent 12-subunit RNA polymerase II. Proc. Natl. Acad. Sci. USA 100, 6964–6968 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brueckner, F., Hennecke, U., Carell, T. & Cramer, P. CPD damage recognition by transcribing RNA polymerase II. Science 315, 859–862 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Broennimann, E.F. et al. The PILATUS 1M detector. J. Synchrotron Radiat. 13, 120–130 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  36. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  PubMed  Google Scholar 

  37. Brunger, A.T. Version 1.2 of the Crystallography and NMR system. Nat. Protocols 2, 2728–2733 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  39. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

  40. Gerber, P.R. & Muller, K. MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry. J. Comput. Aided Mol. Des. 9, 251–268 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Armache, K.-J., Mitterweger, S., Meinhart, A. & Cramer, P. Structures of complete RNA polymerase II and its subcomplex Rpb4/7. J. Biol. Chem. 280, 7131–7134 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Kostrewa and other members of the Cramer laboratory for help. We thank D. Temiakov for discussions. F.B. was supported by the Nanosystems Initiative Munich (NIM) and the Elitenetzwerk Bayern (ENB). P.C. was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich SFB646, the Transregio 5 Chromatin, the EU research grant network 3D Repertoire and the Fonds der Chemischen Industrie. Part of this work was performed at the Swiss Light Source (SLS) at the Paul Scherrer Institut, Villigen, Switzerland.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, Munich, 81377, Germany

    Florian Brueckner & Patrick Cramer

Authors
  1. Florian Brueckner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrick Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patrick Cramer.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brueckner, F., Cramer, P. Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nat Struct Mol Biol 15, 811–818 (2008). https://doi.org/10.1038/nsmb.1458

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1458

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