Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis

Pistol ribozyme (Psr) is a distinctclass of small endonucleolyticribozymes, which are important experimental systems for defining fundamentalprinciples of RNA catalysis and designing valuable tools in biotechnology.High-resolution structures of Psr, extensive structure-functionstudies, and computation support a mechanism involving one or morecatalytic guanosine nucleobases acting as a general base and divalentmetal ion-bound water acting as an acid to catalyze RNA 2 '-O-transphosphorylation. Yet, for a wide range of pH andmetal ion concentrations, the rate of Psr catalysis is too fast tomeasure manually and the reaction steps that limit catalysis are notwell understood. Here, we use stopped-flow fluorescence spectroscopyto evaluate Psr temperature dependence, solvent H/D isotope effects,and divalent metal ion affinity and specificity unconstrained by limitationsdue to fast kinetics. The results show that Psr catalysis is characterizedby small apparent activation enthalpy and entropy changes and minimaltransition state H/D fractionation, suggesting that one or more pre-equilibriumsteps rather than chemistry is rate limiting. Quantitative analysesof divalent ion dependence confirm that metal aquo ion pK (a) correlates with higher rates of catalysis independentof differences in ion binding affinity. However, ambiguity regardingthe rate-limiting step and similar correlation with related attributessuch as ionic radius and hydration free energy complicate a definitivemechanistic interpretation. These new data provide a framework forfurther interrogation of Psr transition state stabilization and showhow thermal instability, metal ion insolubility at optimal pH, andpre-equilibrium steps such as ion binding and folding limit the catalyticpower of Psr suggesting potential strategies for further optimization.

Automated summary

Psr is an important experimental system for defining RNA catalysis and designing valuable tools in biotechnology. The rate of Psr catalysis is too fast to measure manually and the reaction steps that limit catalysis are not well understood.