Substrate Recognition Mechanism of Protein-only RNase P

Abstract

Ribonuclease P (RNase P) is the enzyme responsible for catalyzing the removal of the 5’ leader sequence from precursor transfer RNA (pre-tRNA) during the essential maturation process of transfer RNA (tRNA). While RNase P was first discovered in lower order organisms as an RNA-based ribozyme, recent work has revealed the existence of a protein-only RNase P (PRORP) within Eukaryotes. The existence of these two types of independently evolved RNase P enzymes, provides the rare opportunity to compare convergent evolutionary strategies of RNA- and protein-based catalysts. Previous work revealed that both the proteinaceous and RNA-based enzymes achieve nucleolytic activity through the same general two-metal ion mechanism. However, how PRORP recognizes tRNA substrates and whether this mechanism is similar to RNA-based RNase P is poorly understood. This work investigates the substrate recognition strategy of PRORP. Mutagenesis and in vitro binding and catalytic activity assays identified several residues within the conserved pentatricopeptide repeat (PPR) domain of Arabidopsis PRORP1 which form binding interactions with pre-tRNA. A crystal structure of the PPR domain bound to tRNA is solved. Residues critical for binding are shown to form interactions with conserved regions of tRNA. This mode of RNA recognition by a PPR domain is novel, differing from the established sequence-specific RNA model. Interestingly, RNA-based RNase P uses a similar mechanism to detect tRNAs and this is additional evidence of convergent evolution between RNA and protein-only forms of RNase P. Overall this work identifies the mechanism of PPR domain pre-tRNA recognition, providing a foundation for the elucidating the function and mechanism of other PPR-motif containing proteins. B