A predictive understanding of the mechanisms whereby molecules of RNA, with a limited repertoire of reactive functional groups, can catalyze complex chemical transformations is of immense importance for the design of emerging technology built from synthetic systems such as Hachomoji RNA/DNA that have vast potential for new chemical, biological and medicinal applications. Much of what is known about catalytic RNA mechanisms derives from detailed study of small endonucleolytic ribozymes. The Varkud Satellite (VS) ribozyme catalyzes site-specific cleavage and ligation of RNA. This ribozyme exhibits features unique among known ribozyme classes and major gaps remain in our understanding of its catalytic mechanism and active site architecture. Here, we present an interdisciplinary approach to unveil the mechanism of VS catalysis in unprecedented detail. We combine phosphorothioate substitution studies, precision nucleobase mutation experiments and linear free energy relationship measurements with atomistic molecular simulations, molecular solvation theory-based calculations, and multidimensional ab initio quantum mechanical/molecular mechanical free energy simulations to elucidate mechanism. Through this unprecedented confluence of theory and experiment, we not only unify the existing body of structural and functional data but establish for the first time in any ribozyme system a comprehensive set of elaborate catalytic interactions, including the degree to which protons are transferred to the leaving group and from the nucleophile in the transition state. In addition, we provide evidence for a critical Mg2+ ion in the active site that interacts with the scissile phosphate and anchors the general base guanine in a position for nucleophile activation. In other known nucleolytic ribozymes, this anchoring is mediated by a conserved nucleobase residue that is strikingly absent in VS. This novel role for Mg2+ adds a new trick to the diverse array of known catalytic RNA strategies and establishes the first functional link between the hammerhead and hairpin ribozyme classes that hints at possible underlying evolutionary connections between them.

Theory and experiment converge to define the active site configuration and catalytic mechanism of the largest known nucleolytic ribozyme

2019 Fall National Meeting

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