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Minimal and RNA-Free Rnase P in Aquifex Aeolicus

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Minimal and RNA-Free Rnase P in Aquifex Aeolicus Minimal and RNA-free RNase P in Aquifex aeolicus Astrid I. Nickela, Nadine B. Wäbera, Markus Gößringera, Marcus Lechnera, Uwe Linneb, Ursula Tothc, Walter Rossmanithc, and Roland K. Hartmanna,1 aInstitute of Pharmaceutical Chemistry, Philipps-Universität Marburg, 35037 Marburg, Germany; bFaculty of Chemistry, Mass Spectrometry, Philipps- Universität Marburg, 35032 Marburg, Germany; and cCenter for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria Edited by Sidney Altman, Yale University, New Haven, CT, and approved September 11, 2017 (received for review May 12, 2017) RNase P is an essential tRNA-processing enzyme in all domains of (Fig. 1B). Several protein bands correlating less strongly with life. We identified an unknown type of protein-only RNase P in the RNase P activity were identified as well. The most abundant hyperthermophilic bacterium Aquifex aeolicus: Without an RNA proteins in fractions containing highest RNase P activity (SI Ap- subunit and the smallest of its kind, the 23-kDa polypeptide com- pendix,Figs.S9andS10), as inferred from mass spectrometry, were prises a metallonuclease domain only. The protein has RNase P in the order of decreasing peptide representation: (i) Aq_880, activity in vitro and rescued the growth of Escherichia coli and (ii) glutamine synthetase, (iii) ribosomal protein S2, (iv)PNPase, Saccharomyces cerevisiae strains with inactivations of their more (v) N utilization substance protein B homolog (NusB), and complex and larger endogenous ribonucleoprotein RNase P. Ho- (vi) Aq_707 (with similarity to an Escherichia coli tRNA binding mologs of Aquifex RNase P (HARP) were identified in many Ar- protein of the MnmC family). Furthermore, Aq_880 was the only chaea and some Bacteria, of which all Archaea and most Bacteria protein that was also found in an HIC fraction with low RNase P also encode an RNA-based RNase P; activity of both RNase P forms activity eluting at 0 M (NH4)2SO4 (SI Appendix, Figs. S3 and S10). from the same bacterium or archaeon could be verified in two The presence of ribosomal protein S2 combined with a previously selected cases. Bioinformatic analyses suggest that A. aeolicus described association of RNase P with 30S ribosomal subunits in and related Aquificaceae likely acquired HARP by horizontal gene Bacillus subtilis (14) suggested a possible association of A. aeolicus transfer from an archaeon. RNase P with the ribosome, which, however, could not be confirmed experimentally. protein-only RNase P | Aquifex aeolicus | tRNA processing | HARP The 22.6-kDa protein Aq_880 was recombinantly expressed in E. coli and affinity-purified using a C-terminal His tag. Addition of BIOCHEMISTRY he architectural diversity of RNase P enzymes is unique: In affinity-purified Aq_880 protein to an active A. aeolicus HIC TBacteria, Archaea, and in the nuclei and organelles of many fraction not only boosted RNase P activity, but recombinant Eukarya, RNase P is a complex consisting of a catalytic RNA Aq_880 alone was able to process the precursor tRNAs specifically subunit and a varying number of proteins (one in Bacteria, at least and efficiently at the canonical RNase P cleavage site without re- four in Archaea, and up to 10 in Eukarya) (1, 2). A different type quiring any additional components (Fig. 2A). We further analyzed of RNase P was discovered more recently in human mitochondria whether processing by recombinant Aq_880 generates tRNA with (3) and, subsequently, in land plants and some protists (4, 5). This a5′-phosphate end, as any other previously characterized RNase form, termed proteinaceous or protein-only RNase P (PRORP), P.TLCindeedconfirmeda5′-phosphate at the 5′-terminal G+1 lacks any RNA subunit and consists of one or three (animal mi- residue of tRNAGly processed by recombinant Aq_880 (Fig. 2 B tochondria) protein subunit(s); it is found in most branches of the and C). A contamination of the recombinant Aq_880 preparation eukaryotic phylogenetic tree (6). with endogenous E. coli RNase P was excluded by RT-PCR anal- Bacterial RNase P enzymes identified so far are composed of a ysis and micrococcal nuclease pretreatment (SI Appendix,Figs. ∼400-nt-long catalytic RNA subunit (encoded by rnpB) and a S11 and S12). Since Aq_707 and PNPase (Aq_221) copurified with small protein subunit of ∼14 kDa (encoded by rnpA) (7). How- RNase P activity and peptides matching Aq_707 and Aq_880 were ever, no rnpA and rnpB genes were identified in the genome of Aquifex aeolicus or other Aquificaceae (8–12). The genetic or- Significance ganization of A. aeolicus tRNAs in tandem clusters and as part of ribosomal operons and the detection of tRNAs with canonical RNase P is a tRNA-processing enzyme of unique architectural di- mature 5′-ends in total RNA extracts from A. aeolicus implied versity: either a catalytic RNA plus one or more (up to 10) pro- the existence of a tRNA 5′-maturation activity (9) that was in- teins, or one (or three) unrelated proteins only. We identified yet deed subsequently detected in cell lysates of A. aeolicus (11, 13). another enzyme form in the bacterium Aquifex aeolicus,a23-kDa However, to date, the identity and biochemical composition of protein and the smallest known form of RNase P. Apparently, it RNase P in A. aeolicus has remained enigmatic. was acquired by horizontal gene transfer from Archaea. In some other bacteria and many archaea, it is simultaneously present Results and Discussion with the presumably more ancient RNA-based enzyme form. Here, we pursued a classical biochemical approach to identify the Bacteria with both activities may represent the missing link of RNase P of A. aeolicus. The purification procedure consisted of RNase P evolution, a transition state that had also been once three consecutive chromatographic steps: anion exchange, hydro- traversed by the Aquificaceae, which, however, later lost their phobic interaction, and size exclusion chromatography (AEC, RNA-based RNase P. HIC, and SEC, respectively; Fig. 1A and SI Appendix, Figs. S1–S8). RNase P activity was assayed at all purification steps. To identify Author contributions: A.I.N., N.B.W., M.G., W.R., and R.K.H. designed research; A.I.N., N.B.W., M.G. and U.T. performed research; U.L. contributed new reagents/analytic tools; putative protein components of the enzyme, fractions with low and A.I.N., N.B.W., M.G., M.L., W.R., and R.K.H. analyzed data; M.G., M.L., W.R., and R.K.H. high RNase P activity from different purification steps were wrote the paper. comparatively analyzed by step-gradient SDS/PAGE, and protein The authors declare no conflict of interest. bands correlating with activity (Fig. 1B) were subjected to mass This article is a PNAS Direct Submission. SI Appendix 1 spectrometry. An example is SEC ( ,Fig.S5)fraction To whom correspondence should be addressed. Email: roland.hartmann@staff. B4 displaying maximum activity combined with an enrichment of uni-marburg.de. protein bands 1 and 2 identified as the hypothetical protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Aq_880 and polynucleotide phosphorylase (PNPase), respectively 1073/pnas.1707862114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1707862114 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 A B cell lysate RNase P activity MA10A12 B1 B2 B4 B6 B8 B10 B12 PAA kDa Anion Exchange Chromatography 7% * 7 8% Hydrophobic Interaction Chromatography 250 * 7 * 6 * 7 130 9% 0.4 M eluate 0.2 M eluate 0 M eluate * 7 M 100 2 (HIC ) (HIC ) (HIC ) A * 04 02 0 * 5 L 70 11% D * 7 I 55 Size Exclusion Chromatography * 3 / * 4 13% T 35 O 25 F 15 * 1 SEC SEC SDS-step-PAGE 15% 04 02 10 Fig. 1. Partial purification and identification of A. aeolicus RNase P. (A) Schematic overview of the purification procedure. The applied chromatography steps and methods are shown as open boxes; the cell lysate and column fractions with RNase P activity are indicated as gray boxes. (B) SDS/PAGE analysis of “SEC0.2” fractions with RNase P activity eluting from a SEC column that had been loaded with a “HIC0.2” sample [material eluted from the hydrophobic interaction column at 0.2 M (NH4)2SO4]. Fractions with low (A10, B12), increasing (A12, B1, B2), maximum (B4), and decreasing (B6, B8, B10) RNase P activity were loaded in order of their elution from the column (SI Appendix, Fig. S5). Prominent protein bands correlating with RNase P activity were excised and identified by mass spectrometry as: 1, hypothetical protein Aq_880; 2, polynucleotide phosphorylase (PNPase); 3, uncharacterized protein homologous to RNA pseudouridine synthase from B. subtilis; 4, hypothetical protein Aq_707; 5, Aq_1754/Aq_707; 6, Aq_808/Aq_707; 7, glutamine synthetase; only two of these proteins (1 and 2) were most abundant in fraction B4, which displayed the highest RNase P activity; M, molecular mass marker. −1 identified in band 6 (Fig. 1B) excised from SDS gels, we tested nM substrate were 0.5 min for kcat and 12 nM for Km (Fig. 2E). whether these recombinantly produced A. aeliocus proteins support The two- to threefold decrease of both parameters relative to RNase P activity (SI Appendix,Fig.S13). This was done by adding those of the single turnover might be explained by product re- excess amounts of recombinant Aq_707 or Aq_221 to a HIC0.4 lease limiting the rate of the multiple-turnover reaction. SEC fraction and analyzing processing at a pretRNA concentration of indicated that native and recombinant Aq_880 elute as a ∼420- 33 nM (SI Appendix,Fig.S13A). The rationale was that Aq_707 or kDa complex (SI Appendix, Fig. S15), suggesting that the protein Aq_221, if being part of the RNase P enzyme complex and con- forms homooligomers (e.g., three hexamers or six trimers).
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