# Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

## TL;DR

This study challenges the traditional view of how a specific ribozyme performs its catalytic function by revealing new insights into its mechanism.

## Contribution

The study introduces a new method to precisely measure the catalytic contributions of different factors in the pistol ribozyme.

## Key findings

- Replacing the guanine N1 atom only reduced the reaction rate by 2.7-fold, challenging the role of γ-catalysis.
- Two other catalytic factors (α and δ) play a more significant role in the pistol ribozyme's overall catalysis.
- The approach enables precise scaling of catalytic contributions for a better understanding of ribozyme mechanisms.

## 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.