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...

BackgroundThe Aquificales are a diverse group of thermophilic bacteria that thrive in terrestrial and marine hydrothermal environments. They can be divided into the families Aquificaceae, Desulfurobacteriaceae and Hydrogenothermaceae. Although eleven fully sequenced and assembled genomes are available, only little is known about this taxonomic order in terms of RNA metabolism.ResultsIn this work, we compare the available genomes, extend their protein annotation, identify regulatory sequences, annotate non-coding RNAs (ncRNAs) of known function, predict novel ncRNA candidates, show idiosyncrasies of the genetic decoding machinery, present two different types of transfer-messenger RNAs and variations of the CRISPR systems. Furthermore, we performed a phylogenetic analysis of the Aquificales based on entire genome sequences, and extended this by a classification among all bacteria using 16S rRNA sequences and a set of orthologous proteins.Combining several in silico features (e.g. conserved and stable secondary structures, GC-content, comparison based on multiple genome alignments) with an in vivo dRNA-seq transcriptome analysis of Aquifex aeolicus, we predict roughly 100 novel ncRNA candidates in this bacterium.ConclusionsWe have here re-analyzed the Aquificales, a group of bacteria thriving in extreme environments, sharing the feature of a small, compact genome with a reduced number of protein and ncRNA genes. We present several classical ncRNAs and riboswitch candidates. By combining in silico analysis with dRNA-seq data of A. aeolicus we predict nearly 100 novel ncRNA candidates.

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