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

Abstract

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 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 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-)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 PRORP-substrate interaction is not critically dependent on any of the affected (pro-)Rp oxygens or guanosine 2-amino groups.

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

“…This suggests that the differentially localized PRORP isozymes are not selective for pretRNAs of different organellar origin. The STO cleavage rate constants among the PRORP isozymes are consistent with a previous study using a bacterial pre-tRNA Gly substrate, where the observed rate constants vary between 0.04 and 0.13 s −1 (Pavlova et al 2012). Despite these similarities, our data indicate that there is a subset of pre-tRNAs that are more efficiently processed by specific PRORP isozymes.…”

Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs

“…4B). This proposed recognition mechanism is slightly distinct from that by bacterial RNase P. It was further described that an Rp-phosphothioate modification of the scissile bond has no influence on the cleavage activity by PRORP1, even though RNase P is highly sensitive to the modification [30]. It is therefore likely that PRORP1 catalyzes the pre-tRNA processing in a manner distinct from RNase P. It could thus be assumed that the proteinaceous RNase P, PRORP1, has evolved independently of the molecular mechanism by which RNA-based RNase P cleaves the pre-tRNA in catalysis.…”

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

“…Furthermore, the data suggest that one of the scissile phosphate oxygens of pre-tRNA interacts with bound metal ion(s). In PRORP, the pro-S p oxygen of pre-tRNA is hypothesized to interact with the active site metal ions (23,36), although this needs to be confirmed experimentally, whereas the pro-R p substrate oxygen coordinates a metal ion in RNA-based RNase P (37,38). Modeling of bound substrate suggests that coordination of the pro-S p or pro-R p oxygen affects the position of the metal ion relative to the scissile phosphate, leading to an alteration in the pre-tRNA binding mode.…”

“…PRORP1 is activated by Mg 2ϩ , Mn 2ϩ , Co 2ϩ , and Ni 2ϩ (but not Ca 2ϩ , Zn 2ϩ , or Cd 2ϩ (7,23)). We measured the rate constant for single turnover cleavage (k max ) at pH 6.5 with saturating concentrations of PRORP1 and metal (Co ).…”