Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme

Abstract

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.

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Fig. 1: Schematic illustration of the VS ribozyme catalytic reaction.
Fig. 2: Computational investigation of catalytic strategies adopted by the VS ribozyme.
Fig. 3: Catalytic mechanism of the VS ribozyme.
Fig. 4: An elaborate network of interactions configures the VS ribozyme for proton transfer and transition-state stabilization.
Fig. 5: The hammerhead (HHr), hairpin (HPr) and VS(VSr) ribozymes use distinct strategies to anchor the L-platform.

Data availability

The data that support the findings of this study are available in the Supplementary Information file and from the corresponding authors upon request.

Code availability

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.

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Acknowledgements

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.

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A.G. and B.P.W. contributed equally to this work. A.G. performed the computations and B.P.W. performed the experiments. A.G., B.P.W., J.A.P. and D.M.Y. co-wrote the paper. T.J.G. developed enabling software and provided technical support to various aspects of the computational studies. N.-S.L. synthesized the phosphoramidites and oligonucleotides. S.H. and S.A.B. provided the hachimoji RNA substrate. S.R. characterized the stereochemistry of the phosphorothioate substrates. J.A.P. and D.M.Y. conceived and co-directed all experimental and computational aspects of the work.

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Correspondence to Joseph A. Piccirilli or Darrin M. York.

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Computational methods, experimental methods, Supplementary Figs. 1–10, Supplementary Table 1 and extended discussions.

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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

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