Structure and Function of Protein-only RNase P.

Abstract

Transfer RNA is essential for deciphering the genetic code. Transcribed as a precursor, containing extra nucleotides on its 5’ and 3’ termini, tRNA must undergo several enzymatic processing steps before functioning in protein translation. One of the first steps in tRNA maturation is removal of the 5’ extraneous sequence, which is catalyzed by ribonuclease P (RNase P). In all domains of life RNase P can be found composed of a single catalytic RNA which associates with a varying number of proteins. However, a protein-only RNase P (PRORP) has evolved for functioning within genome containing organelles such as the mitochondrion and chloroplast, and the nucleus of land plants and some protist. This work investigates the molecular function of PRORP. The crystal structure of PRORP1 from Arabidopsis reveals three domains: a pentatricopeptide repeat (PPR) domain tethered to a metallonuclease domain through a structural-zinc binding site. The pH and metal dependence of PRORP1 catalyzed pre-tRNA hydrolysis with wild-type and active site mutants is consistent with PRORP1 using a two-metal ion mechanism with a metal hydroxide nucleophile to catalyze phosphodiester bond hydrolysis. Interestingly, this proposed catalytic mechanism is similar to the RNA-based RNase P mechanism, providing evidence for the mechanistic convergence of two different macromolecules, RNA and protein. Furthermore, examination of the reactivity of the three PRORP isozymes with a number of pre-tRNAs of different organellar origin demonstrates that the plant PRORP isozymes have overlapping substrate specificities but varying cleavage fidelities. Overall this work provides an understanding of the molecular function of PRORP enzymes and lays the foundation for understanding the more complicated human mitochondrial protein-only RNase P.