Kinetic analysis of a minimal RNA substrate as a tool for identification of inhibitors of bacterial Ribonuclease P; an emerging antibiotic target

Alexandra Chamberlain, Tong Huang, and Michael E Harris

Department of Chemistry, University of Florida, Gainesville FL

RNA strand cleavage by hydrolysis is a fundamental chemical reaction in biology and essential for RNA metabolism in all organisms. Ribonuclease P is a ribonucleoprotein enzyme that produces mature tRNA by hydrolysis of the 5’ leader group of pre-tRNA. Inhibition of functional RNAs is a rapidly advancing field in drug discovery, and ribonuclease P is a high value antibiotic target, therefore is the subject of intense interest.  New structures of the bacterial RNase P ES complexes from cryoEM gives us important information about active site interactions. Interactions between the protein subunit and the leader sequence of pre-tRNA promote a conformational change that affects the binding of active site metal ions. However, we lack an understanding of the contribution of individual enzyme-substrate interactions to association, conformational changes, and catalysis. Each of these processes represent avenues for inhibitor development to block RNase P function.  To address this challenge, we developed model RNase P substrates to report on these steps.  Our goal is to engineer a model synthetic RNA substrate designed to be readily modified with fluorescent probes and chemical modifications, and broadly applicable for inhibitor discovery, validation, and detailed mechanistic characterization. Here we report the application of minimal synthetic helical substrate (MH1) and establish a minimal kinetic scheme for its cleavage by RNase P. We show that 2-aminopurine (2AP) residue at N(-2) allows for detection of ES complex formation as well as product formation in steady state fluorescence experiments.  Application of an N(-2) 2AP substrate allows stopped flow fluorescence to be used to detect the kinetics of both binding and catalytic steps of the mechanism.    Using the MH1 substrate we are investigating the roles of G332 and A333 in the RNase P RNA subunit which were recently identified as forming key interactions with the 5’ leader sequence. Modification of MH1 to contain both Cy3 and BHQ-2 modifications permits plate reader measurements of both time resolved and end-point assays, thus enabling high throughput screening. These results establish a powerful experimental tool for analysis of RNase P molecular recognition and demonstrate its application in screening for new inhibitors of this key metabolic enzyme.