Minimal and RNA-free RNase P in Aquifex aeolicus

Abstract

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...

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

“…While M. mazei HARP was less active in vitro than H. volcanii HARP under the tested assay conditions, an inverse correlation was seen in complementation experiments using an E. coli RNase P depletion strain: M. mazei HARP could rescue growth of E. coli with similar efficiency as the type enzyme Aq_880, whereas H. volcanii HARP was unable to do so. We conclude from these findings that the two archaeal HARPs are basically capable of acting as RNases P. On the other hand, the ease with which HARP gene deletions could be obtained in the two archaea and the lack of considerable growth defects of the deletion strains suggests that the RNA‐based RNase P, expressed in all archaea that encode HARPs , is the major archaeal RNase P activity. This notion receives support from our observation that the RNase P RNA subunit gene could not be deleted in H. volcanii and repression of the RNase P RNA gene caused severe defects in tRNA biogenesis .…”

“…Derivatives of plasmid pDG148(S/X) encoding the HARP proteins without and with C‐terminal His‐tag were transformed into E. coli BW for complementation experiments carried out as described previously .…”

“…Aq_880cHis, used as control regarding activity and length of the released 5 0 -flank, was previously demonstrated to cleave the canonical class I precursor tRNA Gly (pre-tRNA Gly , Fig. 5A) at the canonical site (19). Both archaeal HARP proteins also processed this substrate at the canonical site (Fig.…”

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

“…It is noteworthy that in archaea and bacteria, 5' tRNA enzymes containing a similar catalytic domain to PRORPs were also recently identified (HARPs, Homologs of Aquifex RNase P). These enzymes were shown to be the smallest RNase P with independent 5' end pre-tRNA processing endonuclease activity (Nickel et al 2017). In contrast to human mtRNase P, PRORPs are stand-alone enzymes that do not require accessory proteins.…”

Distribution of the ribosome associated endonuclease Rae1 and the potential role of conserved amino acids in codon recognition

“…Only a handful of ribonucleases (RNases) can be considered as pervasive in biology and no single RNA processing or decay enzyme is truly ubiquitous. For a long time, it was assumed that the RNA subunit of the tRNA 5' processing enzyme RNase P was universally conserved, but it is now known that at least three different enzymes can catalyze this reaction, two of which are protein-only forms [1,2]. Some organisms have even found a way to survive without RNase P, by initiating transcription at the first nucleotide (nt) of the mature tRNA [3].…”