Issue 31, 2022

Who stole the proton? Suspect general base guanine found with a smoking gun in the pistol ribozyme

Abstract

The pistol ribozyme (Psr) is one among the most recently discovered classes of small nucleolytic ribozymes that catalyze site-specific RNA self-cleavage through 2′-O-transphosphorylation. The Psr contains a conserved guanine (G40) that in crystal structures is in a position suggesting it plays the role of the general base to abstract a proton from the nucleophile to activate the reaction. Although some functional data is consistent with this mechanistic role, a notable exception is 2-aminopurine (2AP) substitution which has no effect on the rate, unlike similar substitutions across other so-called “G + M” and “G + A” ribozyme classes. Herein we postulate that an alternate conserved guanine, G42, is the primary general base, and provide evidence from molecular simulations that the active site of Psr can undergo local refolding into a structure that is consistent with the common “L-platform/L-scaffold” architecture identified in G + M and G + A ribozyme classes with Psr currently the notable exception. We summarize the key currently available experimental data and present new classical and combined quantum mechanical/molecular mechanical simulation results that collectively suggest a new hypothesis. We hypothesize that there are two available catalytic pathways supported by different conformational states connected by a local refolding of the active site: (1) a primary pathway with an active site architecture aligned with the L-platform/L-scaffold framework where G42 acts as a general base, and (2) a secondary pathway with the crystallographic active site architecture where G40 acts as a general base. We go on to make several experimentally testable predictions, and suggest specific experiments that would ultimately bring closure to the mystery as to “who stole the proton in the pistol ribozyme?”.

Graphical abstract: Who stole the proton? Suspect general base guanine found with a smoking gun in the pistol ribozyme

  • This article is part of the themed collection: New Talent

Supplementary files

Article information

Article type
Paper
Submitted
02 feb 2022
Accepted
13 apr 2022
First published
14 apr 2022

Org. Biomol. Chem., 2022,20, 6219-6230

Who stole the proton? Suspect general base guanine found with a smoking gun in the pistol ribozyme

Ş. Ekesan and D. M. York, Org. Biomol. Chem., 2022, 20, 6219 DOI: 10.1039/D2OB00234E

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