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Confluence of Theory and Experiment Reveal the Catalytic Mechanism of the Varkud Satellite Ribozyme

The Varkud Satellite (VS) ribozyme catalyzes 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...

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Detalles Bibliográficos
Autores principales: Ganguly, Abir, Weissman, Benjamin P., Giese, Timothy J., Li, Nan-Sheng, Hoshika, Shuichi, Rao, Saieesh, Benner, Steven A., Piccirilli, Joseph A., York, Darrin M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389185/
https://www.ncbi.nlm.nih.gov/pubmed/31959957
http://dx.doi.org/10.1038/s41557-019-0391-x
Descripción
Sumario:The Varkud Satellite (VS) ribozyme catalyzes 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 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 transfers in the transition state. Further, we provide evidence for a critical Mg(2+) ion 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 Mg(2+) adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the VS, hairpin, and hammerhead ribozyme classes.