An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

Abstract

A predictive understanding of the mechanisms of RNA cleavage is important for the design of emerging technology built from biological and synthetic molecules that have promise for new biochemical and medicinal applications. Over the past 15 years, RNA cleavage reactions involving 2'-O-transphosphorylation have been discussed using a simplified framework introduced by Breaker that consists of four fundamental catalytic strategies (designated α, β, γ, and δ) that contribute to rate enhancement. As more detailed mechanistic data emerge, there is need for the framework to evolve and keep pace. We develop an ontology for discussion of strategies of enzymes that catalyze RNA cleavage via 2'-O-transphosphorylation that stratifies Breaker's framework into primary (1°), secondary (2°) and tertiary (3°) contributions to enable more precise interpretation of mechanism in the context of structure and bonding. Further, we point out instances where atomic-level changes give rise to changes in more than one catalytic contribution, a phenomenon we refer to as 'functional blurring'. We hope that this ontology will help clarify our conversations and pave the path forward toward a consensus view of these fundamental and fascinating mechanisms. The insight gained will deepen our understanding of RNA cleavage reactions catalyzed by natural protein and RNA enzymes, as well as aid in the design of new engineered DNA and synthetic enzymes.

Molecular dynamics analysis of Mg2+‐dependent cleavage of a pistol ribozyme reveals a fail‐safe secondary ion for catalysis

“…These mechanistic delineations of the cleavage reaction are crucial for understanding the activity of the molecule. Biochemical assays and atomic‐resolution structural models support this simple framework for understanding the mechanism of catalysis …”

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

“…In this work, we perform a series of computational and experimental investigations on the VS ribozyme in order to characterize its functionally active state, identify its key catalytic strategies, and unify the existing body of structural and functional data on this important system. We adopt a recently developed ontology for discussion of RNA cleavage reactions, 23 extended from the original framework introduced by Breaker 24 , specifically, α catalysis, acquirement of in-line nucleophilic attack; β catalysis, stabilization of negative charge on a non-bridge phosphoryl oxygens (NPOs); ɣ catalysis, activation (deprotonation) of the 2’-hydroxyl group to generate the nucleophile; and δ catalysis, stabilization of negative charge on the 5’-oxygen of the leaving group ( Figure 1 ). The results are divided into eight sections.…”

Crucial Roles of Two Hydrated Mg2+ Ions in Reaction Catalysis of the Pistol Ribozyme

“…Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone.T hey are widely distributed in nature and are essential for rolling-circle-based replication of satellite and pathogenic RNAs. [1][2][3][4][5][6][7][8][9][10][11][12][13] Comparative genomic analysis led to the discovery of novel selfcleaving ribozymes,n amed twister,t wister-sister, pistol, and hatchet. [14,15] Forthe first three classes,the three-dimensional architectures [16][17][18][19][20][21][22][23][24][25] in pre-cleavage states were solved by X-ray crystallography,a nd very recently,a lso the first structure of ah atchet ribozyme (product) has been determined.…”