The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg2+ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg2+ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.
This is a preview of subscription content, access via your institution
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available in the Supplementary Information file and from the corresponding authors upon request.
Simulation software are available in the latest release of AMBER18. Example input files, representative structures, animation of the active site in the presence and absence of the Mg2+ ion derived from the MD simulations and an animation of the catalytic reaction derived from the simulations are provided online free to download: http://theory.rutgers.edu.
Abelson, J. The discovery of catalytic RNA. Nat. Rev. Mol. Cell. Biol. 18, 653 (2017).
Symons, R. H. Small catalytic RNAs. Ann. Rev. Biochem. 61, 641–671 (1992).
Herschlag, D. & Cech, T. R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry 29, 10159–10171 (1990).
Narlikar, G. J. & Herschlag, D. Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. Ann. Rev. Biochem. 66, 19–59 (1997).
Hoshika, S. et al. Hachimoji DNA and RNA: a genetic system with eight building blocks. Science 363, 884–887 (2019).
Jimenez, R. M., Polanco, J. A. & Lupták, A. Chemistry and biology of self-cleaving ribozymes. Trends Biochem. Sci. 40, 648–661 (2015).
Wilson, T. J., Liu, Y. & Lilley, D. M. J. Ribozymes and the mechanisms that underlie RNA catalysis. Front. Chem. Sci. Eng. 10, 178–185 (2016).
Kennell, J. C. et al. The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid. Gene Dev. 9, 294–303 (1995).
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).
Lafontaine, D. A., Norman, D. G. & Lilley, D. M. J. The global structure of the VS ribozyme. EMBO J. 21, 2461–2471 (2002).
Lafontaine, D. A., Wilson, T. J., Zhao, Z. Y. & Lilley, D. M. J. Functional group requirements in the probable active site of the VS ribozyme. J. Mol. Biol. 323, 23–34 (2002).
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).
Hiley, S. L., Sood, V. D., Fan, J. & Collins, R. A. 4-thio-U cross-linking identifies the active site of the VS ribozyme. EMBO J. 21, 4691–4698 (2002).
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).
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).
Jaikaran, D., Smith, M. D., Mehdizadeh, R., Olive, J. & Collins, R. A. An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method. RNA 14, 938–949 (2008).
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).
Smith, M. D. & Collins, R. A. Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme. Proc. Natl Acad. Sci. USA 104, 5818–5823 (2007).
Suslov, N. B. et al. Crystal structure of the Varkud satellite ribozyme. Nat. Chem. Biol. 11, 840–846 (2015).
DasGupta, S., Suslov, N. B. & Piccirilli, J. A. Structural basis for substrate helix remodeling and cleavage loop activation in the Varkud satellite ribozyme. J. Am. Chem. Soc. 139, 9591–9597 (2017).
Collins, R. A. The Neurospora Varkud satellite ribozyme. Biochem. Soc. Trans. 30, 1122–1126 (2002).
Lilley, D. M. J. The Varkud satellite ribozyme. RNA 10, 151–158 (2004).
Bevilacqua, P. C. et al. An ontology for facilitating discussion of catalytic strategies of RNA-cleaving enzymes. ACS Chem. Biol. 14, 1068–1076 (2019).
Emilsson, G. M., Nakamura, S., Roth, A. & Breaker, R. R. Ribozyme speed limits. RNA 9, 907–918 (2003).
Kovacheva, Y. S., Tzokov, S. B., Murray, I. A. & Grasby, J. A. The role of phosphate groups in the VS ribozyme–substrate interaction. Nucleic Acids Res. 32, 6240–6250 (2004).
Zamel, R. & Collins, R. A. Rearrangement of substrate secondary structure facilitates binding to the Neurospora VS ribozyme. J. Mol. Biol. 324, 903–915 (2002).
Bingaman, J. L. et al. The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme. Nat. Chem. Biol. 13, 439–445 (2017).
Campbell, D. O. & Legault, P. Nuclear magnetic resonance structure of the Varkud satellite ribozyme stem-loop V RNA and magnesium-ion binding from chemical-shift mapping. Biochemistry 44, 4157–4170 (2005).
Campbell, D. O., Bouchard, P., Desjardins, G. & Legault, P. NMR structure of Varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites. Biochemistry 45, 10591–10605 (2006).
Bonneau, E. & Legault, P. Nuclear magnetic resonance structure of the III–IV–V three-way junction from the Varkud satellite ribozyme and identification of magnesium-binding sites using paramagnetic relaxation enhancement. Biochemistry 53, 6264–6275 (2014).
Bonneau, E. & Legault, P. NMR localization of divalent cations at the active site of the Neurospora VS ribozyme provides insights into RNA–metal-ion interactions. Biochemistry 53, 579–590 (2014).
Dagenais, P., Girard, N., Bonneau, E. & Legault, P. Insights into RNA structure and dynamics from recent NMR and X-ray studies of the Neurospora Varkud satellite ribozyme. WIREs RNA 8, e1421 (2017).
Murray, J. B., Seyhan, A. A., Walter, N. G., Burke, J. M. & Scott, W. G. The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem. Biol. 5, 587–595 (1998).
Maguire, J. L. & Collins, R. A. Effects of cobalt hexammine on folding and self-cleavage of the Neurospora VS ribozyme. J. Mol. Biol. 309, 45–56 (2001).
Tzokov, S. B., Murray, I. A. & Grasby, J. A. The role of magnesium ions and 2′-hydroxyl groups in the VS ribozyme-substrate interaction. J. Mol. Biol. 324, 215–226 (2002).
Luchko, T. et al. Three-dimensional molecular theory of solvation coupled with molecular dynamics in Amber. J. Chem. Theory Comput. 6, 607–624 (2010).
Genheden, S., Luchko, T., Gusarov, S., Kovalenko, A. & Ryde, U. An MM/3D-RISM approach for ligand binding affinities. J. Phys. Chem. B 114, 8505–8516 (2010).
Sood, V. D., Beattie, T. L. & Collins, R. A. Identification of phosphate groups involved in metal binding and tertiary interactions in the core of the Neurospora VS ribozyme. J. Mol. Biol. 282, 741–750 (1998).
Kim, S. H., Bartholomew, D. G., Allen, L. B., Robins, R. K. & Revankar, G. R. Imidazo[1,2-a]-s-triazine nucleosides. Synthesis and antiviral activity of the N-bridgehead guanine, guanosine, and guanosine monophosphate analogues of imidazo[1,2-a]-s-triazine. J. Med. Chem. 21, 883–889 (1978).
Krauch, T. et al. New base-pairs for DNA and RNA. In Abstracts of Papers at the 196th ACS National Meeting of the American Chemical Society (American Chemical Society, 1988).
Georgiadis, M. M. et al. Structural basis for a six nucleotide genetic alphabet. J. Am. Chem. Soc. 137, 6947–6955 (2015).
Jimenez, R. M., Polanco, J. A. & Luptak, A. Chemistry and biology of self-cleaving ribozymes. Trends Biochem. Sci. 40, 648–661 (2015).
Koo, S. C. et al. Transition state features in the hepatitis delta virus ribozyme reaction revealed by atomic perturbations. J. Am. Chem. Soc. 137, 8973–8982 (2015).
Weinan, E., Liu, D. & Vanden-Eijnden, E. Nested stochastic simulation algorithm for chemical kinetic systems with disparate rates. J. Chem. Phys. 123, 194107 (2005).
Vanden-Eijnden, E. & Venturoli, M. Revisiting the finite temperature string method for the calculation of reaction tubes and free energies. J. Chem. Phys. 130, 194103 (2009).
Zamel, R. et al. Exceptionally fast self-cleavage by a Neurospora Varkud satellite ribozyme. Proc. Natl Acad. Sci. USA 101, 1467–1472 (2004).
Mir, A. et al. Two divalent metal ions and conformational changes play roles in the hammerhead ribozyme cleavage reaction. Biochemistry 54, 6369–6381 (2015).
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).
Chen, H., Giese, T. J., Golden, B. L. & York, D. M. Divalent metal ion activation of a guanine general base in the hammerhead ribozyme: insights from molecular simulations. Biochemistry 56, 2985–2994 (2017).
Gaines, C. S., Piccirilli, J. A. & York, D. M. The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design. RNA https://doi.org/10.1261/rna.071894.119 (2019).
Ward, W. L., Plakos, K. & DeRose, V. J. Nucleic acid catalysis: metals, nucleobases, and other cofactors. Chem. Rev. 114, 4318–4342 (2014).
Anderson, M., Schultz, E. P., Martick, M. & Scott, W. G. Active-site monovalent cations revealed in a 1.55-Å-resolution hammerhead ribozyme structure. J. Mol. Biol. 425, 3790–3798 (2013).
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).
We thank S. DasGupta for valuable discussions. A.G., B.W., J.A.P. and D.M.Y. are grateful for the financial support provided by the National Institutes of Health (grant GM62248 to D.M.Y. and grant GM131568 to J.A.P.). B.P.W. acknowledges support from the Predoctoral Training Program in Chemistry and Biology (T32-GM008720). Computational resources were provided by the National Institutes of Health under grant no. S10OD012346, the Office of Advanced Research Computing (OARC) at Rutgers, the State University of New Jersey, Rutgers Discovery Information Institute (RDI2), the State University of New Jersey, and by the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant no. OCI-1053575 (project no. TG-MCB110101). This research is also part of the Blue Waters sustained-petascale computing project, which is supported by the National Science Foundation (awards OCI-0725070 and ACI-1238993) and the state of Illinois. Blue Waters is a joint effort of the University of Illinois at Urbana-Champaign and its National Center for Supercomputing Applications.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Ganguly, A., Weissman, B.P., Giese, T.J. et al. Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme. Nat. Chem. 12, 193–201 (2020). https://doi.org/10.1038/s41557-019-0391-x