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Crystal structure and mechanistic investigation of the twister ribozyme

Abstract

We present a crystal structure at 2.3-Å resolution of the recently described nucleolytic ribozyme twister. The RNA adopts a previously uncharacterized compact fold based on a double-pseudoknot structure, with the active site at its center. Eight highly conserved nucleobases stabilize the core of the ribozyme through the formation of one Watson-Crick and three noncanonical base pairs, and the highly conserved adenine 3′ of the scissile phosphate is bound in the major groove of an adjacent pseudoknot. A strongly conserved guanine nucleobase directs its Watson-Crick edge toward the scissile phosphate in the crystal structure, and mechanistic evidence supports a role for this guanine as either a general base or acid in a concerted, general acid-base–catalyzed cleavage reaction.

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Figure 1: The sequence and structure of the twister ribozyme.
Figure 2: The structural role of conserved nucleotides in the twister ribozyme.
Figure 3: The active center of the twister ribozyme.
Figure 4: The catalytic mechanism of the twister ribozyme.

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References

  1. Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

    CAS  Article  PubMed  Google Scholar 

  2. Fica, S.M. et al. RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Lilley, D.M.J. & Eckstein, F. in Ribozymes and RNA Catalysis (eds. Lilley, D.M.J. & Eckstein, F.) 1–318 (Royal Society of Chemistry, Cambridge, 2008).

  4. Lilley, D.M.J. & Sutherland, J. in The Chemical Origins of Life and its Early Evolution (eds. Lilley, D.M. & Sutherland, J.) 2851–2986 (Royal Society Publishing, London, 2011).

  5. 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).

    CAS  Article  Google Scholar 

  6. Forster, A.C. & Symons, R.H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 49, 211–220 (1987).

    CAS  Article  PubMed  Google Scholar 

  7. Haseloff, J. & Gerlach, W.L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334, 585–591 (1988).

    CAS  Article  PubMed  Google Scholar 

  8. 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).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. de la Peña, M. & Garcia-Robles, I. Intronic hammerhead ribozymes are ultraconserved in the human genome. EMBO Rep. 11, 711–716 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Seehafer, C., Kalweit, A., Steger, G., Graf, S. & Hammann, C. From alpaca to zebrafish: hammerhead ribozymes wherever you look. RNA 17, 21–26 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  13. Wilson, T.J. & Lilley, D.M.J. The evolution of ribozyme chemistry. Science 323, 1436–1438 (2009).

    CAS  Article  PubMed  Google Scholar 

  14. Han, J. & Burke, J.M. Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site. Biochemistry 44, 7864–7870 (2005).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Klein, D.J., Been, M.D. & Ferré-D'Amaré, A.R. Essential role of an active-site guanine in glmS ribozyme catalysis. J. Am. Chem. Soc. 129, 14858–14859 (2007).

    CAS  Article  PubMed  Google Scholar 

  17. Wilson, T.J., McLeod, A.C. & Lilley, D.M.J. A guanine nucleobase important for catalysis by the VS ribozyme. EMBO J. 26, 2489–2500 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Cochrane, J.C., Lipchock, S.V., Smith, K.D. & Strobel, S.A. Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme. Biochemistry 48, 3239–3246 (2009).

    CAS  Article  PubMed  Google Scholar 

  19. Kath-Schorr, S. et al. General acid-base catalysis mediated by nucleobases in the hairpin ribozyme. J. Am. Chem. Soc. 134, 16717–16724 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Lafontaine, D.A., Wilson, T.J., Norman, D.G. & Lilley, D.M.J. The A730 loop is an important component of the active site of the VS ribozyme. J. Mol. Biol. 312, 663–674 (2001).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  22. Wilson, T.J. et al. Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme. Proc. Natl. Acad. Sci. USA 107, 11751–11756 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Wilson, T.J. & Lilley, D.M.J. Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid-base catalysis? A critical assessment of available experimental data. RNA 17, 213–221 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Nakano, S., Chadalavada, D.M. & Bevilacqua, P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science 287, 1493–1497 (2000).

    CAS  Article  PubMed  Google Scholar 

  25. 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).

    CAS  Article  PubMed  Google Scholar 

  26. Das, S.R. & Piccirilli, J.A. General acid catalysis by the hepatitis delta virus ribozyme. Nat. Chem. Biol. 1, 45–52 (2005).

    CAS  Article  PubMed  Google Scholar 

  27. Chen, J.H. et al. A 1.9-Å crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage. Biochemistry 49, 6508–6518 (2010).

    CAS  Article  PubMed  Google Scholar 

  28. Lee, T.S. et al. Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation. J. Am. Chem. Soc. 130, 3053–3064 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Thomas, J.M. & Perrin, D.M. Probing general acid catalysis in the hammerhead ribozyme. J. Am. Chem. Soc. 131, 1135–1143 (2009).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  31. 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).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  33. Bevilacqua, P.C. Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry 42, 2259–2265 (2003).

    CAS  Article  PubMed  Google Scholar 

  34. Ouellet, J., Melcher, S.E., Iqbal, A., Ding, Y. & Lilley, D.M.J. Structure of the three-way helical junction of the hepatitis C virus IRES element. RNA 16, 1597–1609 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Tan, E. et al. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc. Natl. Acad. Sci. USA 100, 9308–9313 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Pley, H.W., Flaherty, K.M. & McKay, D.B. Three-dimensional structure of a hammerhead ribozyme. Nature 372, 68–74 (1994).

    CAS  Article  PubMed  Google Scholar 

  37. Murray, J.B. et al. The structural basis of hammerhead ribozyme self-cleavage. Cell 92, 665–673 (1998).

    CAS  Article  PubMed  Google Scholar 

  38. Lipfert, J., Ouellet, J., Norman, D.G., Doniach, S. & Lilley, D.M.J. The complete VS ribozyme in solution studied by small-angle X-ray scattering. Structure 16, 1357–1367 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  40. Emilsson, G.M., Nakamura, S., Roth, A. & Breaker, R.R. Ribozyme speed limits. RNA 9, 907–918 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Kuzmin, Y.I., Da Costa, C.P., Cottrell, J.W. & Fedor, M.J. Role of an active site adenine in hairpin ribozyme catalysis. J. Mol. Biol. 349, 989–1010 (2005).

    CAS  Article  PubMed  Google Scholar 

  42. Beaucage, S.L. & Caruthers, M.H. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedr. Lett. 22, 1859–1862 (1981).

    CAS  Article  Google Scholar 

  43. Wilson, T.J., Zhao, Z.-Y., Maxwell, K., Kontogiannis, L. & Lilley, D.M.J. Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme. Biochemistry 40, 2291–2302 (2001).

    CAS  Article  PubMed  Google Scholar 

  44. Hakimelahi, G.H., Proba, Z.A. & Ogilvie, K.K. High yield selective 3′-silylation of ribonucleosides. Tetrahedr. Lett. 22, 5243–5246 (1981).

    CAS  Article  Google Scholar 

  45. Perreault, J.-P., Wu, T., Cousineau, B., Ogilvie, K.K. & Cedergren, R. Mixed deoxyribo- and ribooligonucleotides with catalytic activity. Nature 344, 565–567 (1990).

    CAS  Article  PubMed  Google Scholar 

  46. Milligan, J.F., Groebe, D.R., Witherall, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Leslie, A.G.W. & Powell, H.R. Processing diffraction data with Mosflm. in Evolving Methods for Macromolecular Crystallography 245, 41–51 (2007).

    Article  Google Scholar 

  48. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are extremely grateful to R. Breaker for early communication of his data on the twister ribozyme and B. Hunter and L. Huang for valuable advice and discussion. The work was funded by Cancer Research UK (program grant A11722).

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Y.L. performed crystallography; T.J.W. performed mechanistic investigations; S.A.M. synthesized RNA; and Y.L., T.J.W. and D.M.J.L. designed the research, analyzed data and wrote the manuscript.

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Correspondence to David M J Lilley.

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Liu, Y., Wilson, T., McPhee, S. et al. Crystal structure and mechanistic investigation of the twister ribozyme. Nat Chem Biol 10, 739–744 (2014). https://doi.org/10.1038/nchembio.1587

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