Skip to main content

Thank you for visiting 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.

Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate


RNA polymerase II (Pol II) utilizes the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript via its intrinsic nuclease activity to maintain high transcriptional fidelity critical for cell growth and viability. The detailed catalytic mechanism of intrinsic cleavage remains unknown. Here, we combined ab initio quantum mechanics/molecular mechanics studies and biochemical cleavage assays to show that Pol II utilizes downstream phosphate oxygen to activate the attacking nucleophile in hydrolysis, while the newly formed 3′-end is protonated through active-site water without a defined general acid. Experimentally, alteration of downstream phosphate oxygen either by 2′-5′ sugar linkage or stereo-specific thio-substitution of phosphate oxygen drastically reduced cleavage rate. We showed by N7-modification that guanine nucleobase is not directly involved as an acid–base catalyst. Our proposed mechanism provides important insights into the intrinsic transcriptional cleavage reaction, an essential step in transcriptional fidelity control.

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

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Fig. 1: Elucidating the Pol II intrinsic cleavage mechanism using aiQM/MM–MD simulations.
Fig. 2: Identifying reaction pathway using free-energy profiles.
Fig. 3: Structural representation along the reaction pathway.
Fig. 4: Alteration of the RNA 3′-terminal linkage reduces the intrinsic cleavage rate.
Fig. 5: The effect of thio-substitution of non-bridging phosphate oxygen on intrinsic cleavage.

Code availability

Custom computer code used to analyse simulation data is available from the corresponding author on request.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.


  1. Vermulst, M. et al. Transcription errors induce proteotoxic stress and shorten cellular lifespan. Nat. Commun. 6, 8065 (2015).

    Article  CAS  Google Scholar 

  2. Borukhov, S., Polyakov, A., Nikiforov, V. & Goldfarb, A. GreA protein: transcription elongation factor from Escherichia coli. Proc. Natl Acad. Sci. USA 89, 8899–8902 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Reines, D. Elongation factor-dependent transcript shortening by template-engaged RNA polymerase II. J. Biol. Chem. 267, 3795–3800 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lange, U. & Hausner, W. Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS-induced RNA cleavage. Mol. Microbiol. 52, 1133–1143 (2004).

    Article  CAS  Google Scholar 

  6. Sigurdsson, S., Dirac-Svejstrup, A. B. & Svejstrup, J. Q. Evidence that transcript cleavage is essential for RNA polymerase II transcription and cell viability. Mol. Cell 38, 202–210 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Sekine, S., Murayama, Y., Svetlov, V., Nudler, E. & Yokoyama, S. The ratcheted and ratchetable structural states of RNA polymerase underlie multiple transcriptional functions. Mol. Cell 57, 408–421 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Nielsen, S. & Zenkin, N. Transcript assisted phosphodiester bond hydrolysis by eukaryotic RNA polymerase II. Transcription 4, 209–212 (2013).

    Article  Google Scholar 

  11. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993).

    Article  CAS  Google Scholar 

  12. Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y. & Yang, W. Watching DNA polymerase η make a phosphodiester bond. Nature 487, 196–201 (2012).

    Article  CAS  Google Scholar 

  13. Gao, Y. & Yang, W. Capture of a third Mg2+ is essential for catalyzing DNA synthesis. Science 352, 1334–1337 (2016).

    Article  CAS  Google Scholar 

  14. Freudenthal, B. D., Beard, W. A., Shock, D. D. & Wilson, S. H. Observing a DNA polymerase choose right from wrong. Cell 154, 157–168 (2013).

    Article  CAS  Google Scholar 

  15. Freudenthal, B. D. et al. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature 517, 635–639 (2015).

    Article  CAS  Google Scholar 

  16. Vyas, R., Reed, A. J., Tokarsky, E. J. & Suo, Z. Viewing human DNA polymerase β faithfully and unfaithfully bypass an oxidative lesion by time-dependent crystallography. J. Am. Chem. Soc. 137, 5225–5230 (2015).

    Article  CAS  Google Scholar 

  17. Reed, A. J. & Suo, Z. Time-dependent extension from an 8-oxoguanine lesion by human DNA polymerase beta. J. Am. Chem. Soc. 139, 9684–9690 (2017).

    Article  CAS  Google Scholar 

  18. Jamsen, J. A. et al. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action. Nat. Commun. 8, 253 (2017).

    Article  Google Scholar 

  19. Basu, R. S. & Murakami, K. S. Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze x-ray crystallography. J. Biol. Chem. 288, 3305–3311 (2013).

    Article  CAS  Google Scholar 

  20. Molina, R. et al. Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nat. Struct. Mol. Biol. 22, 65–72 (2014).

    Article  Google Scholar 

  21. Sheng, G. et al. Structure-based cleavage mechanism of Thermus thermophilus argonaute DNA guide strand-mediated DNA target cleavage. Proc. Natl Acad. Sci. USA 111, 652–657 (2014).

    Article  CAS  Google Scholar 

  22. Samara, N. L. & Yang, W. Cation trafficking propels RNA hydrolysis. Nat. Struct. Mol. Biol. 25, 715–721 (2018).

    Article  CAS  Google Scholar 

  23. Yuzenkova, Y. & Zenkin, N. Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis. Proc. Natl Acad. Sci. USA 107, 10878–10883 (2010).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. Mishanina, T. V., Palo, M. Z., Nayak, D., Mooney, R. A. & Landick, R. Trigger loop of RNA polymerase is a positional, not acid-base, catalyst for both transcription and proofreading. Proc. Natl Acad. Sci. USA 114, E5103–E5112 (2017).

    CAS  PubMed  Google Scholar 

  26. Warshel, A. & Levitt, M. Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. J. Mol. Biol. 103, 227–249 (1976).

    Article  CAS  Google Scholar 

  27. Warshel, A. Computer simulations of enzyme catalysis: methods, progress, and insights. Annu. Rev. Biophys. Biomol. Struct. 32, 425–443 (2003).

    Article  CAS  Google Scholar 

  28. Riccardi, D. et al. Development of effective quantum mechanical/molecular mechanical (QM/MM) methods for complex biological processes. J. Phys. Chem. B 110, 6458–6469 (2006).

    Article  CAS  Google Scholar 

  29. Lu, X. et al. QM/MM free energy simulations: recent progress and challenges. Mol. Simul. 42, 1056–1078 (2016).

    Article  CAS  Google Scholar 

  30. Zhou, Y., Wang, S., Li, Y. & Zhang, Y. Born–Oppenheimer ab initio QM/MM molecular dynamics simulations of enzyme reactions. Methods Enzymol. 577, 105–118 (2016).

    Article  CAS  Google Scholar 

  31. Hu, P., Wang, S. & Zhang, Y. How do SET-domain protein lysine methyltransferases achieve the methylation state specificity? Revisited by Ab initio QM/MM molecular dynamics simulations. J. Am. Chem. Soc. 130, 3806–3813 (2008).

    Article  CAS  Google Scholar 

  32. Hu, P., Wang, S. & Zhang, Y. Highly dissociative and concerted mechanism for the nicotinamide cleavage reaction in Sir2Tm enzyme suggested by ab initio QM/MM molecular dynamics simulations. J. Am. Chem. Soc. 130, 16721–16728 (2008).

    Article  CAS  Google Scholar 

  33. Kumar, S., Rosenberg, J. M., Bouzida, D., Swendsen, R. H. & Kollman, P. A. The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem. 13, 1011–1021 (1992).

    Article  CAS  Google Scholar 

  34. Da, L.-T. et al. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue. Nat. Commun. 7, 11244 (2016).

    Article  CAS  Google Scholar 

  35. Gottesman, M. E. & Mustaev, A. Inorganic phosphate, arsenate, and vanadate enhance exonuclease transcript cleavage by RNA polymerase by 2000-fold. Proc. Natl Acad. Sci. USA 115, 2746–2751 (2018).

    Article  CAS  Google Scholar 

  36. Patel, N. et al. Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends. Nucleic Acids Res. 40, 4507–4519 (2012).

    Article  CAS  Google Scholar 

  37. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  38. Anandakrishnan, R., Aguilar, B. & Onufriev, A. V. H++ 3.0: Automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res. 40, W537–W541 (2012).

    Article  CAS  Google Scholar 

  39. Søndergaard, C. R., Olsson, M. H. M., Rostkowski, M. & Jensen, J. H. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J. Chem. Theory Comput. 7, 2284–2295 (2011).

    Article  Google Scholar 

  40. Huang, X. et al. RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription. Proc. Natl Acad. Sci. USA 107, 15745–15750 (2010).

    Article  CAS  Google Scholar 

  41. Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098–3100 (1988).

    Article  CAS  Google Scholar 

  42. Lee, Yang & Parr Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  43. Becke, A. D. Density‐functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  44. Petersson, G. A. et al. A complete basis set model chemistry. I. The total energies of closed‐shell atoms and hydrides of the first‐row elements. J. Chem. Phys. 89, 2193–2218 (1988).

    Article  CAS  Google Scholar 

  45. Petersson, G. A. & Al‐Laham, M. A. A complete basis set model chemistry. II. Open‐shell systems and the total energies of the first‐row atoms. J. Chem. Phys. 94, 6081–6090 (1991).

    Article  CAS  Google Scholar 

  46. Dijkstra, E. W. A note on two problems in connexion with graphs. Numer. Math. 1, 269–271 (1959).

    Article  Google Scholar 

  47. Cui, Q. & Elstner, M. Density functional tight binding: values of semi-empirical methods in an ab initio era. Phys. Chem. Chem. Phys. 16, 14368–14377 (2014).

    Article  CAS  Google Scholar 

Download references


We thank Z. Lin for helpful discussions. This work was supported by the Hong Kong Research Grant Council (grant nos. HKUST C6009-15G and AoE/P-705/16 to X.H. and X.L.; 16302214 and T31-605/18-W to X.H.), the King Abdullah University of Science and Technology Office of Sponsored Research (OSR) (OSR-2016-CRG5-3007 to X.H. and X.G.), the Shenzhen Science and Technology Innovation Committee (JCYJ20170413173837121 to X.H.), the Innovation and Technology Commission (ITC-CNERC14SC01 to X.H.), and the National Institutes of Health (grant no. R35-GM127040 to Y.Z.; grant no. GM102362 to D.W.). X.H. is the Padma Harilela Associate Professor of Science. This research made use of the computing resources of the Supercomputing Laboratory at King Abdullah University of Science and Technology.

Author information

Authors and Affiliations



J.X. and X.L prepared the proteins and performed the biochemical analyses. C.K.M.T., X.G. and S.W. performed aiQM/MM–MD simulations. H.Y.C. and X.L. performed reverse phase-FPLC purification of thio-substituted oligonucleotides. C.K.M.T., J.X., P.P-H.C., F.K.S., D.W., Y.Z., and X.H. analysed the data. C.K.M.T., J.X., P.P-H.C., D.W., Y.Z., and X.H. wrote the manuscript with inputs from all authors. D.W., Y.Z. and X.H. directed and supervised the research.

Corresponding authors

Correspondence to Peter Pak-Hang Cheung, Dong Wang, Yingkai Zhang or Xuhui Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Notes 1–13, Supplementary Figures 1–19, Supplementary References.

Supplementary Data 1

Initial model.

Supplementary Data 2

RS structure.

Supplementary Data 3

TS1 structure.

Supplementary Data 4

IS structure.

Supplementary Data 5

TS2 structure.

Supplementary Data 6

PS structure.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tse, C.K.M., Xu, J., Xu, L. et al. Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate. Nat Catal 2, 228–235 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


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