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:

Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage

Nature Chemical Biology volume 12, pages 702–708 (2016)Cite this article

Subjects

Abstract

The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2′-O of G for attack on the adjacent to-be-cleaved P-O5′ bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2′-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry.

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: Secondary and tertiary structure of the env25 pistol ribozyme.
Figure 2: Structural details of the env25 pistol ribozyme in the vicinity of the G53-U54 cleavage site.
Figure 3: In-line alignment at the G53-U54 cleavage site in the env25 pistol ribozyme and comparison with the structure of the RzB hammerhead ribozyme.
Figure 4: Self-cleavage of the env25 pistol ribozyme.
Figure 5: NMR spectroscopic analysis of the env25 pistol ribozyme in solution.
Figure 6: Cleavage kinetics of the env25 pistol ribozyme analyzed by Ap fluorescence.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Jimenez, R.M., Polanco, J.A. & Luptak, A. Chemistry and biology of self-cleaving ribozymes. Trends Biochem. Sci. 40, 648–661 (2015).

    Article  CAS  Google Scholar 

  2. Weinberg, Z. et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat. Chem. Biol. 11, 606–610 (2015).

    Article  CAS  Google Scholar 

  3. Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, I.R. & Bruening, G. Autolytic processing of dimeric plant virus satellite RNA. Science 231, 1577–1580 (1986).

    Article  CAS  Google Scholar 

  4. Hutchins, C.J., Rathjen, P.D., Forster, A.C. & Symons, R.H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627–3640 (1986).

    Article  CAS  Google Scholar 

  5. Saville, B.J. & Collins, R.A. A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria. Cell 61, 685–696 (1990).

    Article  CAS  Google Scholar 

  6. Perreault, J. et al. Identification of hammerhead ribozymes in all domains of life reveals novel structural variations. PLOS Comput. Biol. 7, e1002031 (2011).

    Article  CAS  Google Scholar 

  7. Webb, C.H., Riccitelli, N.J., Ruminski, D.J. & Luptak, A. Widespread occurrence of self-cleaving ribozymes. Science 326, 953 (2009).

    Article  CAS  Google Scholar 

  8. Buzayan, J.M., Gerlach, W.L. & Bruening, G. Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA. Nature 323, 349–353 (1986).

    Article  CAS  Google Scholar 

  9. Fedor, M.J. Structure and function of the hairpin ribozyme. J. Mol. Biol. 297, 269–291 (2000).

    Article  CAS  Google Scholar 

  10. Winkler, W.C., Nahvi, A., Roth, A., Collins, J.A. & Breaker, R.R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004).

    Article  CAS  Google Scholar 

  11. Watson, P.Y. & Fedor, M.J. The ydaO motif is an ATP-sensing riboswitch in Bacillus subtilis. Nat. Chem. Biol. 8, 963–965 (2012).

    Article  CAS  Google Scholar 

  12. Webb, C.H. & Luptak, A. HDV-like self-cleaving ribozymes. RNA Biol. 8, 719–727 (2011).

    Article  CAS  Google Scholar 

  13. Lilley, D.M. The Varkud satellite ribozyme. RNA 10, 151–158 (2004).

    Article  CAS  Google Scholar 

  14. Martick, M. & Scott, W.G. Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126, 309–320 (2006).

    Article  CAS  Google Scholar 

  15. Rupert, P.B. & Ferre-D'Amare, A.R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature 410, 780–786 (2001).

    Article  CAS  Google Scholar 

  16. Rupert, P.B., Massey, A.P., Sigurdsson, S.T. & Ferre-D'Amare, A.R. Transition state stabilization by a catalytic RNA. Science 298, 1421–1424 (2002).

    Article  CAS  Google Scholar 

  17. Klein, D.J. & Ferre-D'Amare, A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313, 1752–1756 (2006).

    Article  CAS  Google Scholar 

  18. Cochrane, J.C., Lipchock, S.V. & Strobel, S.A. Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor. Chem. Biol. 14, 97–105 (2007).

    Article  CAS  Google Scholar 

  19. Ferré-D'Amaré, A.R., Zhou, K. & Doudna, J.A. Crystal structure of a hepatitis delta virus ribozyme. Nature 395, 567–574 (1998).

    Article  Google Scholar 

  20. Ke, A., Zhou, K., Ding, F., Cate, J.H. & Doudna, J.A. A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature 429, 201–205 (2004).

    Article  CAS  Google Scholar 

  21. Suslov, N.B. et al. Crystal structure of the Varkud satellite ribozyme. Nat. Chem. Biol. 11, 840–846 (2015).

    Article  CAS  Google Scholar 

  22. Doherty, E.A. & Doudna, J.A. Ribozyme structures and mechanisms. Annu. Rev. Biochem. 69, 597–615 (2000).

    Article  CAS  Google Scholar 

  23. Cochrane, J.C. & Strobel, S.A. Catalytic strategies of self-cleaving ribozymes. Acc. Chem. Res. 41, 1027–1035 (2008).

    Article  CAS  Google Scholar 

  24. Ferré-D'Amaré, A.R. & Scott, W.G. Small self-cleaving ribozymes. Cold Spring Harb. Perspect. Biol. 2, a003574 (2010).

    Article  Google Scholar 

  25. Lilley, D.M. Mechanisms of RNA catalysis. Phil. Trans. R. Soc. Lond. B 366, 2910–2917 (2011).

    Article  CAS  Google Scholar 

  26. Roth, A. et al. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat. Chem. Biol. 10, 56–60 (2014).

    Article  CAS  Google Scholar 

  27. Eiler, D., Wang, J. & Steitz, T.A. Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proc. Natl. Acad. Sci. USA 111, 13028–13033 (2014).

    Article  CAS  Google Scholar 

  28. Liu, Y., Wilson, T.J., McPhee, S.A. & Lilley, D.M. Crystal structure and mechanistic investigation of the twister ribozyme. Nat. Chem. Biol. 10, 739–744 (2014).

    Article  CAS  Google Scholar 

  29. Ren, A. et al. In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme. Nat. Commun. 5, 5534 (2014).

    Article  CAS  Google Scholar 

  30. Košutić, M. et al. A mini-twister variant and impact of residues/cations on the phosphodiester cleavage of this ribozyme class. Angew. Chem. Int. Edn. Engl. 54, 15128–15133 (2015).

    Article  Google Scholar 

  31. Gaines, C.S. & York, D.M. Ribozyme catalysis with a twist: active state of the twister ribozyme in solution predicted from molecular simulation. J. Am. Chem. Soc. 138, 3058–3065 (2016).

    Article  CAS  Google Scholar 

  32. Harris, K.A., Lunse, C.E., Li, S., Brewer, K.I. & Breaker, R.R. Biochemical analysis of pistol self-cleaving ribozymes. RNA 21, 1852–1858 (2015).

    Article  CAS  Google Scholar 

  33. Cai, Z. & Tinoco, I. Jr. Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite. Biochemistry 35, 6026–6036 (1996).

    Article  CAS  Google Scholar 

  34. Kirk, S.R., Luedtke, N.W. & Tor, Y. 2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes. Bioorg. Med. Chem. 9, 2295–2301 (2001).

    Article  CAS  Google Scholar 

  35. Jeong, S., Sefcikova, J., Tinsley, R.A., Rueda, D. & Walter, N.G. Trans-acting hepatitis delta virus ribozyme: catalytic core and global structure are dependent on the 5′ substrate sequence. Biochemistry 42, 7727–7740 (2003).

    Article  CAS  Google Scholar 

  36. Kapinos, L.E., Operschall, B.P., Larsen, E. & Sigel, H. Understanding the acid-base properties of adenosine: the intrinsic basicities of N1, N3 and N7. Chemistry 17, 8156–8164 (2011).

    Article  CAS  Google Scholar 

  37. Mir, A. et al. Two divalent metal ions and conformational changes play roles in the hammerhead ribozyme cleavage reaction. Biochemistry 54, 6369–6381 (2015).

    Article  CAS  Google Scholar 

  38. Mir, A. & Golden, B.L. Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue. Biochemistry 55, 633–636 (2016).

    Article  CAS  Google Scholar 

  39. Erlacher, M.D. et al. Efficient ribosomal peptidyl transfer critically relies on the presence of the ribose 2′-OH at A2451 of 23S rRNA. J. Am. Chem. Soc. 128, 4453–4459 (2006).

    Article  CAS  Google Scholar 

  40. Wunderlich, C. Advanced stable isotope labeling for NMR of RNA. PhD thesis, Leopold-Franzens University, Innsbruck, Austria (2015).

  41. Schneider, T.P.T.R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).

    Article  Google Scholar 

  42. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

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

    Article  Google Scholar 

  44. Wilcox, J.L. & Bevilacqua, P.C. pKa shifting in double-stranded RNA is highly dependent upon nearest neighbors and bulge positioning. Biochemistry 52, 7470–7476 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge assistance by staff at Northeastern Collaborative Access Team (NE-CAT) beamlines at the Advanced Photon Source and the experimental station 14-1 at the Stanford Synchrotron Radiation Light Source. X-ray diffraction studies were conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the US National Institutes of Health (NIH). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract DE-AC02-06CH11357. A.R. was supported in part by new faculty start-up funds from Zhejiang University and the Thousand Young Talents Plan of China. The research was supported by US NIH grant 1 U19 CA179564 to D.J.P., by the Memorial Sloan-Kettering Cancer Center Core Grant (P30 CA008748), and the Austrian Science Fund FWF (P27947, I1040 to R.M., P26550, P28725 to C.K.), and the Swiss National Foundation SNF (Early Postdoc.Mobility to J.G.).

Author information

Author notes

  1. Aiming Ren and Nikola Vušurović: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Institute, Zhejiang University, Hangzhou, China

    Aiming Ren

  2. Structural Biology Program, Memorial Sloan-Kettering Cancer, New York, New York, USA

    Aiming Ren, Pu Gao & Dinshaw J Patel

  3. Institute of Organic Chemistry, Leopold Franzens University, Innsbruck, Austria

    Nikola Vušurović, Jennifer Gebetsberger, Michael Juen, Christoph Kreutz & Ronald Micura

  4. Center of Molecular Biosciences (CMBI), Innsbruck, Austria

    Nikola Vušurović, Jennifer Gebetsberger, Michael Juen, Christoph Kreutz & Ronald Micura

Authors
  1. Aiming Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nikola Vušurović
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer Gebetsberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pu Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Juen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christoph Kreutz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ronald Micura
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dinshaw J Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R. undertook all of the crystallographic experiments and the structure analysis with the assistance of P.G. under the supervision of D.J.P. while N.V., J.G. and M.J. were involved in nucleoside phosphoramidite synthesis, RNA preparation and cleavage assays under the supervision of R.M. C.K. designed and performed NMR spectroscopy measurements. The paper was written jointly by A.R., N.V., C.K., R.M. and D.J.P. with input from the remaining authors.

Corresponding authors

Correspondence to Ronald Micura or Dinshaw J Patel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–5. (PDF 2825 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ren, A., Vušurović, N., Gebetsberger, J. et al. Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage. Nat Chem Biol 12, 702–708 (2016). https://doi.org/10.1038/nchembio.2125

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2125

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