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Comparative Study
. 1999 Jul 6;96(14):7803-8.
doi: 10.1073/pnas.96.14.7803.

RNase P RNAs from some Archaea are catalytically active

J A Pannucci  1 , E S Haas, T A Hall, J K Harris, J W Brown
Affiliations
Comparative Study

RNase P RNAs from some Archaea are catalytically active

J A Pannucci et al. Proc Natl Acad Sci U S A. .

Abstract

The RNA subunits of RNase Ps of Archaea and eukaryotes have been thought to depend fundamentally on protein for activity, unlike those of Bacteria that are capable of efficient catalysis in the absence of protein. Although the eukaryotic RNase P RNAs are quite different than those of Bacteria in both sequence and structure, the archaeal RNAs generally contain the sequences and structures of the bacterial, phylogenetically conserved catalytic core. A spectrum of archaeal RNase P RNAs were therefore tested for activity in a wide range of conditions. Many remain inactive in ionically extreme conditions, but catalytic activity could be detected from those of the methanobacteria, thermococci, and halobacteria. Chimeric holoenzymes, reconstituted from the Methanobacterium RNase P RNA and the Bacillus subtilis RNase P protein subunits, were functional at low ionic strength. The properties of the archaeal RNase P RNAs (high ionic-strength requirement, low affinity for substrate, and catalytic reconstitution by bacterial RNase P protein) are similar to synthetic RNase P RNAs that contain all of the catalytic core of the bacterial RNA but lack phylogenetically variable, stabilizing elements.

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Figures

Figure 1
Figure 1
Secondary structures of the M. thermoautotrophicum strain ΔH, M. jannaschii, and Escherichia coli RNase P RNAs (refs. and , and J.K.H., E.S.H., and J.W.B., unpublished data). Helices are numbered P1–P18 as described (24). Helices P4 and P6 are shown with lines and brackets. The 5′ and 3′ ends of the native archaeal RNAs were determined by primer extension and nuclease S1 protection. Additional RNase P RNA sequences and structures are available on the Ribonuclease P database at www.mbio.ncsu.edu/RNaseP/home.html (25).
Figure 2
Figure 2
Catalytic activity of synthetic archaeal RNase P RNAs. Assays contained 4 M ammonium acetate, 300 mM MgCl2, 50 mM Tris⋅Cl (pH 8), 0.1% SDS, 0.05% Nonidet P-40, 1.5 nM uniformly labeled B. subtilis pre-tRNAAsp, and ≈300 nM RNase P RNA (synthesized from cloned genes by transcription in vitro) and were incubated for 3.5 h at 45°C. The location of substrate and product bands are indicated. RNase P RNAs tested were no RNA (NR), E. coli (Eco), M. sedula (Mse), S. acidocaldarius (Sac), P. furiosus (Pfu), T. celer (Tce), M. jannaschii (Mja), M. thermolithotrophicus (Mth), Methanococcus marapaludis (Mma), Methanococcus vannielii (Mva), M. formicicum (Mfo), M. thermoautotrophicum ER-H (ERH), M. thermoautotrophicum Marburg (MtM), M. thermoautotrophicum ER-E (ERE), M. thermoautotrophicum ΔH (MtΔ), M. barkeri (Mba), H. volcanii (Hvo), and Natronobacterium gregoryi (Ngr). Activity by the N. gregoryi RNA is evident but weak. The phylogenetic relationships between these organisms based on small subunit ribosomal RNA sequences (26) are shown as a cladogram above the gel. Placement of the ER-E and ER-H RNAs, which were cloned from enrichment cultures inoculated with wastewater sludge, are based on similarity to the M. thermoautotrophicum strains ΔH and Marburg RNase P RNA sequences.
Figure 3
Figure 3
Oligonucleotide-directed RNase H depletion of RNase P activity. RNase P RNAs were annealed with oligonucleotides complementary to J11/12 in either the M. thermoautotrophicum (MtΔ) or the E. coli (Eco) RNase P RNA and then digested with ribonuclease H. Reagents included in each reaction are indicated above each lane. Activity by the M. thermoautotrophicum RNase P RNA was reduced only in reactions containing the Methanobacterium-specific oligonucleotide; reactions lacking the oligonucleotide or containing the E. coli-specific oligonucleotide were not inhibited. Activity by the E. coli RNase P RNA was reduced only in reactions containing the E. coli-specific oligonucleotide; reactions lacking the oligonucleotide or containing the Methanobacterium-specific oligonucleotide were not inhibited.
Figure 4
Figure 4
Intramolecular cleavage by a unimolecular substrate:enzyme RNA. (A) The secondary structure of the conjugated B. subtilis pre-tRNAAsp:M. formicicum (circularly permuted) RNase P RNA (pre-tpRNA) is shown diagrammatically. (B) Unreacted pre-tpRNA is shown in the first lane. Lanes 2 and 3 are the same RNA, incubated in parallel, at 1 and 100 nM; the extent of cleavage is ≈65% in both cases. The three other weaker cleavage products representing either Mg2+-sensitive sites or miscleavages were not characterized. Lanes 4 and 5 are the trans-cleavage analogs of lanes 1 and 3, in which the B. subtilis pre-tRNAAsp, but not the M. formicicum RNase P RNA, is radiolabeled.
Figure 5
Figure 5
Activity of the M. thermoautotrophicum RNase P RNA as functions of ammonium acetate, MgCl2, and substrate concentration. (A) Activity of the M. thermoautotrophicum RNase P RNA as functions of ammonium acetate and MgCl2 concentration. Reactions contained 20 nM substrate RNA and 1 nM RNase P RNA. Maximal activity in this assay represents 47% cleavage. (B) Activity of the M. thermoautotrophicum RNase P RNA as a function of substrate concentration. Reactions were performed under optimal conditions (see Materials and Methods and A). Reactions contained 10 nM RNase P RNA and were incubated for 2.5 h; points above the dashed horizontal line represent multiple turnovers over the course of the incubation. All data points in both A and B are represented by reactions with less than 50% cleavage, within the linear range of enzyme concentration and time for the reaction (data not shown).
Figure 6
Figure 6
Reconstitution of active chimeric holoenzymes. RNase P RNAs from E. coli (Eco, 38 μg/ml), H. volcanii (Hvo, 170 μg/ml), M. formicicum (Mfo, 60 μg/ml), or M. thermoautotrophicum ΔH (MtΔ, 33 μg/ml) were assayed for RNase P activity in the presence of 0, 0.1, 1, or 10 μg/ml B. subtilis RNase P protein (increasing protein concentration indicated by black wedges above the reaction lanes). Specific RNase P cleavage products are indicated by black arrowheads. The E. coli RNase P RNA is somewhat active by itself under these conditions, but activity is enhanced by the inclusion of the B. subtilis RNase P protein. The M. thermoautotrophicum and M. formicicum RNase P RNAs are not active in the absence of protein under these conditions but were activated by inclusion of the B. subtilis RNase P protein. Correct processing by the H. volcanii RNase P RNA was not observed under these conditions in either the presence or absence of the B. subtilis RNase P protein. However, a specific inappropriate cleavage was generated at the highest concentration of protein; the nature of these products and the apparent miscleavage have not been determined.
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