Mitochondrial genomes are punctuated with tRNAs, making processing of tRNAs an essential step not only for tRNA maturation, but also for the maturation of other RNA species critical for mitochondrial biogenesis. Approximately 2/3 of pathogenic mutations in the human mitochondrial genome lie in sequences encoding tRNAs, underscoring the importance of correct processing and functionality of tRNAs. Ribonuclease P (RNase P) catalyzes tRNA 5' end maturation in all domains of life. Until recently, all known RNase P enzymes were ribonucleoproteins possessing a catalytic RNA component. The discovery of a proteinaceous RNase P from human mitochondria and Arabidopsis thaliana chloroplast and mitochondria has dramatically shifted this paradigm; this RNase P represents a new class of metallonucleases (PROteinacous RNase P (PRORP))1, 2. Nuclear-encoded PRORP is conserved among higher eukaryotes, having evolved recently since yeast mitochondrial genomes encode RNase P with a catalytic RNA. Here we report the crystal structure of PRORP1 at 1.98 � resolution revealing a prototypical metallonuclease domain with conserved aspartate residues important for catalysis, a pentatricopeptide repeat (PPR) domain, and a structural zinc binding site that tethers the two domains together. The metallonuclease domain is the first high resolution structure of a NYN domain (N4BP1, YacP-like Nuclease), a member of the PIN (PilT N-terminal) domain-like fold super-family which includes PIN and FLAP/5' ? 3' nuclease families3. Our biochemical results indicate that PRORP1, like the ribozyme RNase P, is a magnesium-dependent enzyme. The biochemical data also shows that the PPR domain enhances pre-tRNA binding affinity and substrate specificity. These results provide a foundation for understanding RNA maturation in mitochondria and chloroplast by providing insight into the structure, evolution, substrate recognition, and catalytic mechanism of the newly discovered class of PRORP proteins. The nuclease component of human mitochondrial RNase P (MRPP3) shares significant sequence similarity with PRORP1, and our structure can be used as a template for rationalizing human diseases linked to mitochondrial tRNA processing. Furthermore, these studies allow for the direct comparison of the catalytic strategies employed by the only known naturally evolved protein and RNA-based catalysts that perform the same reaction. Figure 1. <img src='https://acs-archive-assets2.s3.amazonaws.com/240nm/uploads/46188_figure1.jpg' alt='' /> 1. Holzmann, J. et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell 135, 462-536 (2008).2. Gobert, A. et al. A single Arabidopsis organellar protein has RNase P activity. Nature structural & molecular biology 17, 740-744 (2010).3. Anantharaman, V. & Aravind, L. Research Paper The NYN Domains. RNA Biology 3 (2006).

Structure of proteinaceous RNase P provides insight into precusor-tRNA processing in mitochondria

244th National Meeting (2012)

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