tRNA Processing by Protein-Only versus RNA-Based RNase P: Kinetic Analysis Reveals Mechanistic Differences

In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5'-end maturation, is carried out by three homologous polypeptides (proteinaceous RNase P (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (kreact/Km(sto)) of 3 X 106 M-1min-1 was determined under single-turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an Rp-phosphorothioate modification at the canonical cleavage site in a 5'-precursor tRNA substrate; whereas processing by bacterial RNase P is inhibited by three orders of magnitude in the presence of this sulfur substitution and Mg2+ as the metal-ion cofactor, the PRORP enzymes are affected by not more than a factor of five under the same conditions, without significantly increased miscleavage. These findings indicate that the catalytic mechanism utilized by proteinaceous RNase P is different from that of RNA-based bacterial RNase P, taking place without a direct metal-ion coordination to the (pro-)Rp substituent. As Rp-phosphorothioate and inosine modification at all 26 G residues of the tRNA body had only minor effects on processing by PRORP, we conclude that productive PRORPsubstrate interaction is not critically dependent on any of the affected (pro-)Rp oxygens or guanosine 2-amino groups.