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.

Keywords: Aquifex aeolicus; HARP; protein-only RNase P; tRNA processing.

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 “SEC_0.2” fractions with RNase P activity eluting from a SEC column that had been loaded with a “HIC_0.2” 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.

Fig. 2.

(A) Processing of Thermus thermophilus pretRNA^Gly by the “HIC_0.4” fraction [material eluted from a HIC column at 0.4 M (NH₄)₂SO₄; Center], by recombinant Aq_880 (final concentration 0.1 µg/µL) (Right), or by a mixture of both (Left); Bsu, B. subtilis RNase P as positive control; S, the 5′-[³²P]-endlabeled pretRNA^Gly substrate; L, the 5′-leader cleavage product. (B and C) Analysis of the 5′-end of pretRNA^Gly as processed by recombinant Aq_880 in vitro. PretRNA^Gly was labeled by in vitro transcription in the presence of [α-³²P]GTP and processed with recombinant Aq_880 under standard conditions to nearly completeness. The 5′-mature tRNA product was gel-purified, and a part of the eluate was treated with alkaline phosphatase. Untreated (B) and phosphatase-treated (C) RNAs were subjected to alkaline hydrolysis, and monophosphate and diphosphate nucleosides were resolved by 2D TLC. The pGp bisphosphate (B) is sensitive to phosphatase pretreatment (C) consistent with its origin from the tRNA’s 5′-end. Spot intensities correspond to the number of the respective nucleosides that are followed by a G in the tRNA sequence and, thereby, are radioactively labeled (A, 8; C, 8; G, 9; U, 1). (D) Single-turnover kinetics of recombinant Aq_880 for the processing of pretRNA^Gly (data points are based on three to six independent determinations for each concentration of Aq_880); error bars are SDs. Kinetic constants and the standard errors of the curve fit are k_react = 1.43 ± 0.14 min⁻¹ and K_m(sto) = 33 ± 8 nM. (E) Multiple-turnover kinetics of pretRNA^Gly processing using 1 nM Aq_880. Results are based on three independent determinations for each pretRNA concentration. Error bars are SDs of the mean. Kinetic constants and the standard errors of the curve fit are k_cat = 0.52 ± 0.04 min⁻¹ and K_m = 12 ± 4 nM. (F) Thermostability of recombinant Aq_880 (gray bars) or a HIC_0.4 fraction containing native RNase P (black bars) in comparison with the E. coli RNase P holoenzyme (white and hatched bars, respectively). RNase P activities were preincubated either at 37 °C or 85 °C before their catalytic activity was determined under single-turnover conditions at 37 °C. Error bars are standard deviations of the mean based on at least three experiments. Note that the activity of E. coli RNase P after preincubation at 85 °C was so low that the corresponding bar to the right of the black HIC_0.4 bar does not rise above the baseline.

Fig. 3.

Complementation of E. coli and yeast RNase P by Aq_880. (A) A. aeolicus Aq_880 is able to functionally replace bacterial RNase P in vivo. E. coli BW cells transformed with expression plasmids for Aq_880 or its variants D138A, D142A, D144A, and D160A were grown at 37 °C under permissive (in the presence of arabinose; +ara) or nonpermissive (in the presence of glucose; +glu) conditions. Already after 1 d, cell growth was detectable in E. coli BW cells containing the expression vector for Aq_880; whereas no complementation was observed with the aspartate to alanine mutants D138A, D142A, and D160A, weak colony formation was seen with D144A. rnpB: E. coli RNase P as positive control. (−), the empty vector as negative control. (B) Expression and solubility of Aq_880 variants in E. coli BW. Soluble (s) and insoluble (is) protein fractions from E. coli BW expressing the Aq_880 variants were analyzed by Western blotting using a polyclonal antiserum to Aq_880. No signal was obtained when E. coli BW was transformed with the empty vector (−) or E. coli rnpB (+), respectively; molecular mass marker (in kilodaltons) indicated on the left. (C) Aq_880 and its variant D144A rescue growth upon deletion of the yeast nuclear RNase P RNA gene RPR1. Two colonies each obtained through rescue by Aq_880 or its variant D144A were applied as spots in 10-fold serial dilution to YPD plates, and the growth of the strains was monitored in parallel to a control (rescue by RPR1). Note the later appearance and smaller colony size of Aq_880 and D144A strains indicating slow growth. (D) Genotyping of Aq_880 and D144A colonies derived from plasmid shuffle. The analysis of a control (rpr1Δ::kanMX4 [RPR1]) and two complementation isolates each (Aq_880, genotype rpr1Δ::kanMX4 [Aq_880]; D144A, genotype rpr1Δ::kanMX4 [Aq_880^D144A]) is shown. The deletion of RPR1, the integrity of the chromosomal gene disruption, and the presence of Aq_880 were verified by PCR. The part of the gene interrogated by the genotyping PCR is indicated by a black bar on top of the gene cartoon (RPR1, blue; kanMX4, yellow; Aq_880, magenta; promoter/terminator regions, gray) to the right of each agarose gel panel.

Fig. 4.

Phylogenetic distribution of HARP. Condensed phylogenetic tree of HARPs. Bacteria were condensed at order level, Archaea at phylum level. Bootstrap values are indicated at the branches (inferred from 1,000 replicates). Filled and empty circles indicate the presence or absence, respectively, of RNase P RNA (RNA subunit), the bacterial RNase P protein (RnpA), or the archaeal RNase P protein subunits Pop5/Rpp30; half-filled circles indicate that we identified for (at least some) bacterial species in this order only an Rpp30 (white/black circles) or Pop5 homolog (black/white circle), but not both.