Catalysis by an "RNA-free" RNase P: Structure and Function of MRPP3, the Presumptive Nuclease Subunit of Human Mitochondrial RNase P

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5` ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized until just recently are composed of a catalytic RNA plus one or more proteins, and tRNA 5` end maturation was thus generally considered a universal ribozyme-catalyzed process. We recently overcame this paradigm when we identified the components of human mitochondrial RNase P (mtRNase P), finding only proteins, and reconstituted the enzymatic activity using just three of these. Evidently, a complex of three proteins replaced the ribozyme-remnant from the hypothetical RNA world during the evolution of animal mitochondria. This protein enzyme nonetheless drives the same biochemical reaction and fulfills the same biological function as its RNA-based predecessor. The RNase P family appears thus unique in biology as it includes isoforms representing partial as well as complete evolutionary transitions from RNA- to protein-based catalysis. In this project we aim to elucidate the catalytic mechanism of mtRNase P, and to compare the catalytic strategy of this protein enzyme to that of its more common ribozymal isoforms. mtRNase P is composed of three proteins, termed MRPP1-3, at least two of which are involved in other tRNA- related and -unrelated biochemical pathways too. MRPP1 and MRPP2 constitute a methyltransferase, involved in the modification of mitochondrial tRNAs. They also appear to confer tRNA-specificity to mtRNase P, but only upon addition of the third protein, MRPP3, and magnesium, tRNA 5` end cleavage can be reconstituted. MRPP3 contains putative RNA-binding and metallonuclease domains and it is thus reasonable to assume that it is the actual nuclease moiety of the enzyme. By determining (1) the atomic scale structure of MRPP3, its molecular interactions with the other components of mtRNase P and with tRNA precursors, by dissecting (2) functional groups of enzyme and substrate involved in substrate recognition, cleavage site positioning, and catalysis, by studying (3) the role and interactions of metal ions involved in hydrolysis, and by studying (4) the cell biology and evolution of MRPP3, we aim to obtain a comprehensive understanding of the catalytic mechanism and biology of mtRNase P. Ultimately, we may learn how and why evolution replaced an ancient RNA catalyst in the animal mitochondrial lineage, while it retained ribozyme catalysis in the vast majority of phylogenetic domains.

Ribonuclease P (RNase P) is the enzyme that removes extensions from the 5 end of transfer RNA (tRNA) precursors. Despite this rather simple enzymatic role, the enzyme family is one of the most diverse known. The ubiquitous enzyme was originally identified as a presumably ancient RNA enzyme (ribozyme) with a protein moiety that during evolution increased from 1 in Bacteria to 10 in the nucleus of Eukarya. In contrast to these ribonucleoproteins, we previously found that human mitochondrial RNase P is a protein complex without any RNA subunit. In the course of this project we now showed that a protein rather than an RNA is in fact responsible for RNase P function in the mitochondria and chloroplasts, but also in the nucleus of many more Eukarya. In striking contrast to the complex ribonucleoprotein enzymes previously found in the nucleus, this form of RNase P is a simple, single-subunit protein. Although the RNA and protein forms of RNase P use a somewhat different catalytic mechanism, their substrate specificities and functional properties are astonishingly similar, and the enzyme forms could even be exchanged one for the other without significantly impairing organismal fitness. RNase P thereby represents a unique and extreme case of convergent evolution at the molecular level.