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

Abstract

Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N −1 of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10 4 -10 5 M −1 s −1 ) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3 ′ -discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

Minimal and RNA-free RNase P in Aquifex aeolicus

“…2D) were determined as k react = 1.43 min −1 and K M(sto) = 33 nM, thus similar to the values previously obtained for protein-only RNase P (PRORP3) from Arabidopsis thaliana [k react = 1.7 min (15) and similar to activities determined by others (16). The multiple-turnover kinetic parameters determined under the same conditions at 1 nM enzyme and 5-200 nM substrate were 0.5 min −1 for k cat and 12 nM for K m (Fig.…”

Determination of protein‐only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease

“…For example, it might trim long leader sequences, while PRORP1 would perform the final maturation of shortened leader sequences. This would be in agreement with the observations that pre‐tRNAs are often transcribed with long leader sequences in plant mitochondria (Hammani and Giegé, ), while in vitro PRORP cleaves pre‐tRNAs with very short leader sequences with higher efficiency (Howard et al ., ). Such a cooperation between two nucleases for RNA maturation would be reminiscent of the concerted action of RNase II and PNPase for the 3′ end maturation of mRNAs in plant mitochondria (Perrin et al ., ; Stoll and Binder, ).…”

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

“…In contrast to human mtRNase P, PRORPs are stand-alone enzymes that do not require accessory proteins. Most studies of protein-based RNase Ps have focused on PRORPs because these enzymes are homologous to MRPP3 and provide a tractable system for study (Pinker et al 2017;Gobert et al 2013;Gutmann, Gobert, and Giege 2012;Gobert et al 2010;Karasik et al 2016;Howard et al 2013;Howard et al 2012;Howard et al 2016;Klemm et al 2016). As a consequence, little is known about how the components of the metazoan mtRNase P complex function.…”