The (nearly) ubiquitous enzyme is found in two fundamentally different forms: (i) as a complex of an ancient, structurally conserved RNA molecule with a variable number of proteins (1 in Bacteria, 4–5 in Archaea, up to 10 in Eukarya), where the RNA is the actual catalyst and active alone under specific in vitro conditions ( 3–7) (ii) as a ∼60-kDa protein called PRORP (proteinaceous or protein-only RNase P), which does not contain a nucleic acid as enzyme subunit, although it requires additional proteins in some cases ( 8–12) PRORP is found in various Eukarya, but not in Bacteria or Archaea. RNase P is the endonuclease responsible for the removal of transcriptional 5′-leader sequences from tRNA precursors (pre-tRNAs) ( 1, 2). Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5′ or 3′ extensions of the tRNA. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. RNase P is the enzyme that removes 5′ extensions from tRNA precursors.
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