Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

Abstract

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

Assessment of Nucleobase Protomeric and Tautomeric States in Nucleic Acid Structures for Interaction Analysis and Structure-Based Ligand Design

“…This deviates from the proposed reaction mechanism which requires N1 as an acceptor for the 2′-OH hydrogen which can be provided by G either via its enol tautomer (G*) or deprotonation (G − ). 30,31,71,78 The hydrogen atom of 2′-OH is oriented toward the phosphate of the following residue, G6. While this differs from the common ribozyme catalysis mechanism, several aspects must be considered.…”