The endoribonuclease RNase P is responsible for tRNA 5\' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (homolog of Aquifex RNase P). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene rnpB) and protein (gene rnpA) subunits of RNP RNase P, but only the HARP of the thermophile Thermodesulfatator indicus (phylum Thermodesulfobacteria) was found to have robust tRNA 5\'-end maturation activity in vitro and in vivo in an Escherichia coli RNase P depletion strain. These findings suggest that both types of RNase P are able to contribute to the essential tRNA 5\'-end maturation activity in T. indicus, thus resembling the predicted evolutionary transition state in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes in this family of bacteria. Remarkably, T. indicus RNase P RNA is transcribed with a P12 expansion segment that is posttranscriptionally excised in vivo, such that the major fraction of the RNA is fragmented and thereby truncated by ∼70 nt in the native T. indicus host as well as in the E. coli complementation strain. Replacing the native P12 element of T. indicus RNase P RNA with the short P12 helix of Thermotoga maritima RNase P RNA abolished fragmentation, but simultaneously impaired complementation efficiency in E. coli cells, suggesting that intracellular fragmentation and truncation of T. indicus RNase P RNA may be beneficial to RNA folding and/or enzymatic activity.

RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon.

The maturation of tRNA precursors involves the 5\' cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes.