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 ( k react / K m(sto) ) of 3×10 6   M −1  min −1 was determined under single‐turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an R p‐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 Mg 2+ 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‐) R p substituent. As R p‐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 PRORP–substrate interaction is not critically dependent on any of the affected (pro‐) R p oxygens or guanosine 2‐amino groups.