Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

Abstract

Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.catalytic mechanism | magnesium | molecular recognition A ccording to the RNA world hypothesis, RNA played dual roles as carrier of genetic information and catalyst in a prebiotic world. However, over eons of evolution, proteins with their expanded 20-aa alphabet and greater structural and functional complexity took over many RNA-based functions. Structural insights into this evolutionary transition are limited because of the lack of examples of RNA and protein macromolecules that perform the same biological function in nature. One notable exception is ribonuclease P (RNase P) that catalyzes maturation of the 5′ end of tRNA across all domains of life. Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P [proteinacous RNase P (PRORP)] from human mitochondria and Arabidopsis thaliana has dramatically shifted this paradigm (1-3). These enzymes represent a unique class of metallonucleases and are conserved among many eukaroytes (1, 2). A. thaliana encodes three PRORP enzymes (PRORP1, -2, and -3) that catalyze pretRNA processing (2). PRORP1 localizes to the mitochondria and chloroplast whereas PRORP2 and PRORP3 localize to the nucleus (2), suggesting that protein-based enzymes catalyze pretRNA maturation in these cellular locations (2, 3). To gain mechanistic and evolutionary insights into the PRORP...

RNase P enzymes

“…16 None of the domains have homology to any of the protein components of eukaryotic or bacterial RNase P enzymes, consistent with bioinformatic data suggesting their disparate evolution. The PPR domain enhances the affinity for pre-tRNA binding and is proposed to play an important role in orienting the pre-tRNA substrate for cleavage.…”

“…18 Our structure of A. thaliana PRORP1 revealed an active site that can bind two manganese atoms via conserved aspartate residues. 16 This observation led us to propose a two-metal ion catalytic mechanism, similar to those previously proposed for RNA-based RNase P and members of the FLAP nuclease family. Despite the two-metal ion similarity with RNA-based RNase P, we propose that active site amino acid chains act as general acid/base catalysts in PRORP enzymes.…”

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

“…The structure of A. thaliana PRORP1 revealed three domains: a Nedd4-BP1, YacP nuclease (NYN) metallonuclease domain, a central structural zinc-binding domain, and a pentatricopeptide repeat domain involved in pre-tRNA binding (Fig. 1A) (7). The NYN domain is a novel metallonuclease domain sharing structural homology to the PIN (PilT N terminus) and flap nuclease families (8).…”

“…The NYN domain is a novel metallonuclease domain sharing structural homology to the PIN (PilT N terminus) and flap nuclease families (8). NYN domains have a relatively exposed active site and contain four conserved aspartates as compared with the flap nuclease family that has six conserved aspartates (7,8). Despite having only four conserved potential metal ligands within its active site, the crystal structure visualized the binding of two manganese ions to the NYN domain of PRORP1 in positions similar to those observed in the flap nuclease family (7,9).…”

An artificial PPR scaffold for programmable RNA recognition

“…PPR proteins contain a repeated motif that is typically 35 amino acids in length and folds into two anti-parallel alpha helices [11][12][13][14] . Natural proteins have been observed to contain between two and thirty individual PPRs 15 .…”