Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

Abstract

We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH3, 2'-H or 2'-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2'-substituent at nt -1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3' -endo ribose and without the H-bond donor function of the 2'-substituent. LNA and 2'-OCH3 suppressed processing at the major aberrant m-1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2'-OCH3 supports a model where the extra methylene (LNA) or methyl group (2'-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects. © The Author(s) 2009. Published by Oxford University Press.