Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site.

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2'-deoxy, 2'-amino, and 2'-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5'-direction. Our findings are in line with the 2'-OH substituent at nucleotide -1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.