RNA strand cleavage by hydrolysis is prevalent in RNA metabolism and affects everything from bacterial persistence to human translation. Due to the importance of this class of reactions, enzymatic and non-enzymatic RNA strand cleavage reactions have been extensively studied revealing a range of catalytic mechanisms and transition states. Ribonuclease P is a ribonucleoprotein that produces mature tRNA by hydrolysis of the 5’ leader group of pre-tRNA. Ribonuclease P is a possible antibiotic target, and therefore has been extensively studied, and new structures of the bacterial RNase P ES complex from cryoEM gives us important information about active site interactions. However, precise mechanistic and details regarding transition state interactions are not yet known, and these are the details that we hope to elucidate. Here we show the characterization of model RNase P substrates designed to answer these mechanistic questions.  Minimal RNase P substrates will be used to measure kinetic isotope effects which will provide a powerful way to experimentally characterize transition states. Kinetic isotope effects in conjuncture with computational simulations of alternative mechanistic scenarios will be used to trim the mechanistic tree until we are left with a singular model or models of the RNase P transition state.  Recent KIE studies in our lab showed that the cleavage of RNA by proteins and ribozymes can stabilize different transition states, yet few experimental systems have been investigated at this level of mechanistic detail. This mechanistic detail can be used to more intelligently design inhibitors as well as broaden our basic understanding of how enzymes catalyze reactions.

Kinetic isotope effects is a tool to illuminate mechanistic details of Ribonuclease P catalysis

ACS Fall 2021

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