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.protein-only RNase P | Aquifex aeolicus | tRNA processing | HARP T he architectural diversity of RNase P enzymes is unique: In Bacteria, Archaea, and in the nuclei and organelles of many Eukarya, RNase P is a complex consisting of a catalytic RNA subunit and a varying number of proteins (one in Bacteria, at least four in Archaea, and up to 10 in Eukarya) (1, 2). A different type of RNase P was discovered more recently in human mitochondria (3) and, subsequently, in land plants and some protists (4, 5). This form, termed proteinaceous or protein-only RNase P (PRORP), lacks any RNA subunit and consists of one or three (animal mitochondria) protein subunit(s); it is found in most branches of the eukaryotic phylogenetic tree (6).Bacterial RNase P enzymes identified so far are composed of a ∼400-nt-long catalytic RNA subunit (encoded by rnpB) and a small protein subunit of ∼14 kDa (encoded by rnpA) (7). However, no rnpA and rnpB genes were identified in the genome of Aquifex aeolicus or other Aquificaceae (8-12). The genetic organization of A. aeolicus tRNAs in tandem clusters and as part of ribosomal operons and the detection of tRNAs with canonical mature 5′-ends in total RNA extracts from A. aeolicus implied the existence of a tRNA 5′-maturation activity (9) that was indeed subsequently detected in cell lysates of A. aeolicus (11, 13). However, to date, the identity and biochemical composition of RNase P in A. aeolicus has remained enigmatic. Results and DiscussionHere, we pursued a classical biochemical approach to identify the RNase P of A. aeolicus. The purification procedure consisted of three consecutive chromatographic steps: anion exchange, hydrophobic interaction, and size exclusion chromatography (AEC, HIC, and SEC, respectively; Fig. 1A and SI Appendix, Figs. S1-S8). RNase P activity was assayed at all purification steps. To identify putative protein components of the enzyme, fractions with low and high RNase P activity from different purification steps were comparatively analyzed by step-gradient SDS/PAGE, and protein bands correlating with activity (Fig. 1B) were subjected to mass spectrometry. An example i...

The mature 5′‐ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein‐only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA‐based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5′‐end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5′‐end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109–1116, 2019

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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 post-transcriptionally 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.

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