Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology

Abstract

The catalytic properties of RNA have been a subject of fascination and intense research since their discovery over 30 years ago. Very recently, several classes of nucleolytic ribozymes have emerged and been characterized structurally. Among these, the twister ribozyme has been center-stage, and a topic of debate about its architecture and mechanism owing to conflicting interpretations of different crystal structures, and in some cases conflicting interpretations of the same functional data. In the present work, we attempt to clean up the mechanistic "debris" generated by twister ribozymes using a comprehensive computational RNA enzymology approach aimed to provide a unified interpretation of existing structural and functional data. Simulations in the crystalline environment and in solution provide insight into the origins of observed differences in crystal structures, and coalesce on a common active site architecture, and dynamical ensemble in solution. We use GPU-accelerated free energy methods with enhanced sampling to ascertain microscopic nucleobase pK a values of the implicated general acid and base, from which predicted activity-pH profiles can be compared directly with experiments. Next, ab initio quantum mechanical/molecular mechanical (QM/MM) simulations with full dynamic solvation under periodic boundary conditions are used to determine mechanistic pathways through multi-dimensional free energy landscapes for the reaction. We then characterize the rate-controlling transition state, and make predictions about kinetic isotope effects and linear free energy relations. Computational mutagenesis is performed to explain the origin of rate effects caused by chemical modifications and make experimentally testable predictions. Finally, we provide evidence that helps to resolve conflicting issues related to the role of metal ions in catalysis. Throughout each stage, we highlight how a conserved L-platform structural motif, to-gether with a key L-anchor residue, forms the characteristic active site scaffold enabling each of the catalytic strategies to come together not only for the twister ribozyme, but the majority of the known small nucleolytic ribozyme classes.

Classification of the nucleolytic ribozymes based upon catalytic mechanism

“…The active site shows how evolution has generated a perfect environment for catalysis of the transesterification, comprising in-line attack, transition state stabilization, and concerted general acid–base catalysis ( Figure 3). An alternative mechanism has been proposed for the twister ribozyme; for a neutral perspective on this, the reader is directed to an excellent critical evaluation by Breaker 39 and a very recent computational study 40 .…”

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

“…However, the field lacks a comprehensive understanding of the influence of solvated divalent cations on catalysis. Prior work on several self‐cleaving RNAs has shed light on the necessity of magnesium ions for maintaining structural integrity, as well as directly participating in catalysis . Recent structural and mechanistic work on pistol ribozymes highlights the unique paradigm of catalysis where a solvated divalent ion is crucial for involvement as a general acid .…”

Mg2+Impacts the Twister Ribozyme through Push-Pull Stabilization of Non-Sequential Phosphate Pairs

“…This suggests the Mg 2+ is specifically interacting with the RNA to stabilize the conformation of this region thereby facilitating formation of the T1 contact upon full folding of the RNA. Previous studies have reported conflicting results on the importance of this stem in folding and activity (34,37,38). Interestingly, Mg 2+ is not seen in this region of Twister in the crystallographic structures (34,38).…”

“…The Twister ribozyme was selected for the present study based on the availability of a range of structural and biochemical data (33)(34)(35)(36)(37)(38). While Twister sequences are extremely widespread, the crystal structure (PDB 4OJI) of the ribozyme used in this study is based on Osa-1-4 sequence from Oryza Sativa (34).…”