Scaling Catalytic Contributions of Small Self-Cleaving Ribozymes

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 (gamma-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 gamma-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that gamma-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (alpha and delta). Our approach allows scaling of the different catalytic contributions (alpha, beta, gamma, delta) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

Automated summary

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. However, the general base knockout study on pistol ribozymes challenges the common view that γ-catalysis critically depends on the N1 moiety. The overall catalysis of pistol ribozymes is made up by three catalytic factors (α, β, γ).