Escherichia coli RNase P RNA: Substrate Ribose Modifications at G+1, but Not Nucleotide − 1/+73 Base Pairing, Affect the Transition State for Cleavage Chemistry

The temperature dependence of processing of precursor tRNAGly (ptRNAGly) variants carrying a single 2′-OCH3 or locked nucleic acid (LNA) modification at G+1 by Escherichia coli endoribonuclease P RNA was studied at rate-limiting chemistry. We show, for the first time, that these ribose modifications at nucleotide + 1 increase the activation energy and alter the activation parameters for the transition state of hydrolysis at the canonical (c0) cleavage site (between nucleotides − 1 and + 1). The modified substrates, particularly the one with LNA at G+1, caused an increase in the activation enthalpy ΔH‡, which was partly compensated for by a simultaneous increase in the activation entropy ΔS‡. NMR imino proton spectra of model acceptor stems derived from the same ptRNA variants unveiled that a riboT or U at − 1 forms two hydrogen bonds with U+73, thus extending the acceptor stem by 1 bp. The non-canonical base pair is substantially stabilized by LNA substitution at nucleotides − 1 or + 1. To address if the activation energy increase owing to LNA at G+1 stems from dissociation of the U(− 1)–U(+ 73) base pair as a prerequisite for interaction of U(+ 73) with U294 in endoribonuclease P RNA, we tested a ptRNAGly variant that is capable of forming an extra C(− 1)–G(+ 73) Watson–Crick base pair. However, compared with a control ptRNA (C at − 1, U at + 73), no significant change in activation parameters was observed for this ptRNA. Thus, our results argue against the possibility that breaking of an additional base pair at the end of the acceptor stem may present an energetic barrier for reaching the transition state of the chemical step for cleavage at the canonical (c0) phosphodiester.