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NameAlexandra Chamberlain
Organization or InstitutionUniversity of Florida
TopicBiochemistry / Chem Bio.
Title

Rapid kinetic analysis of Escherichia coli RNase P active site interactions using minimal substrate containing an intrinsic florescent probe

Author(s)

Alexandra Chamberlain, Tong Huang, and Michael E Harris

Author Institution(s)

Department of Chemistry, University of Florida, Gainesville FL

Abstract

RNA strand cleavage by hydrolysis is prevalent in RNA metabolism and affects everything from bacterial persistence to human translation. 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. 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. To address this challenge, we developed model RNase P substrates to report on these steps that are designed to be used in rapid kinetic experiments.  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.  Incorporation of a Cy3 fluorophore at the 5’ end of MH1 permits the binding step to be confirmed using fluorescence anisotropy.  Using the dual fluorescence MH1 substrate we are investigating the roles of A248, G332 and A333 in the RNase P RNA subunit that were recently identified as forming key interactions with the 5’ leader sequence. These results establish a powerful experimental tool for analysis of RNase P molecular recognition and demonstrate its application in testing key predictions of current models of the RNase P-ptRNA complex.