Characterization of RNA-based and protein-only RNases P from bacteria encoding both enzyme types

Abstract

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (homolog of Aquifex RNase P). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene rnpB) and protein (gene rnpA) subunits of RNP RNase P, but only the HARP of the thermophile Thermodesulfatator indicus (phylum Thermodesulfobacteria) was found to have robust tRNA 5′-end maturation activity in vitro and in vivo in an Escherichia coli RNase P depletion strain. These findings suggest that both types of RNase P are able to contribute to the essential tRNA 5′-end maturation activity in T. indicus, thus resembling the predicted evolutionary transition state in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes in this family of bacteria. Remarkably, T. indicus RNase P RNA is transcribed with a P12 expansion segment that is posttranscriptionally excised in vivo, such that the major fraction of the RNA is fragmented and thereby truncated by ∼70 nt in the native T. indicus host as well as in the E. coli complementation strain. Replacing the native P12 element of T. indicus RNase P RNA with the short P12 helix of Thermotoga maritima RNase P RNA abolished fragmentation, but simultaneously impaired complementation efficiency in E. coli cells, suggesting that intracellular fragmentation and truncation of T. indicus RNase P RNA may be beneficial to RNA folding and/or enzymatic activity.

INTRODUCTION

Transfer RNAs (tRNAs) are transcribed as precursor molecules with extra sequences at their 5′- and 3′-ends that need to be removed for their functionality as adapter molecules in cellular protein synthesis. 5′-end maturation is generally catalyzed by the essential endonuclease RNase P that occurs in ribonucleoprotein (RNP) and protein-only forms. The bacterial RNP enzyme, present in the vast majority of bacteria and considered to be the ancient form of RNase P, is composed of a catalytic RNA component (P RNA, ∼340 to 400 nt) and a small protein cofactor (P or RnpA protein, ∼13 kDa) that is essential for activity in vivo (for review, see Schencking et al. 2020). A small group of bacteria, such as Aquifex aeolicus and its relatives within the family Aquificaceae, lost the RNP enzyme in evolution and replaced it with a single polypeptide of ∼23 kDa (Nickel et al. 2017). This type of protein-only RNase P, termed HARP for homolog of Aquifex RNase P, belongs to the PIN_5 cluster (VapC structural group) within the superfamily of PIN domain-like metallonucleases (Matelska et al. 2017). HARPs form homo-oligomeric structures up to dodecamers, with tetramers proposed to represent the minimal catalytic unit (Feyh et al. 2021; Teramoto et al. 2021; Li et al. 2022). HARP monomers consist of the metallonuclease domain into which a small helical domain is inserted. These small helical domains of two monomers associate to form a helix bundle, termed spike-helix (SH; Feyh et al. 2021) or protruding helix (PrH; Teramoto et al. 2021), resulting in dimers as the basic functional unit of HARP enzymes. For pre-tRNA binding, one dimer interacts with the tRNA T loop region, while the scissile phosphodiester bond at the 5′-end of the acceptor stem reaches into the active site of a metallonuclease domain provided by a neighboring HARP dimer. The conformation of HARPs in the absence of (pre-)tRNA (Feyh et al. 2021; Teramoto et al. 2021; Li et al. 2022) differs from the conformation of a HARP dimer in complex with a pre-tRNA (Li et al. 2022), suggesting conformational changes upon enzyme-substrate complex formation. It is thus unclear at present which oligomeric states are catalytically active. This includes the possibility that dodecamers represent inactive (pre-)tRNA storage forms. Bioinformatic analyses revealed the sporadic occurrence of HARPs in a few Proteobacteria, in Thermodesulfobacteria, Nitrospirae, Verrucomicrobia, Planctomycetes, and in some unclassified bacteria (Nickel et al. 2017; Daniels et al. 2019). Several of those bacteria encode a HARP in addition to the RNP enzyme. HARPs were found to be even more widespread in archaea where they coexist with RNP RNase P. A study on two HARP-expressing euryarchaeotes (Schwarz et al. 2019), Haloferax volcanii and Methanosarcina mazei, revealed tRNA 5′-end maturation activity of their HARP enzymes. However, deletion of the single harp genes caused no growth defect, whereas reduced expression of canonical RNase P RNA resulted in severe tRNA processing and growth defects in H. volcanii (Stachler and Marchfelder 2016). These findings led to the conclusion that archaeal HARPs are not major contributors to global tRNA 5′-end maturation in archaea, but may well exert specialized, yet unknown functions in (t)RNA metabolism (Schwarz et al. 2019).

A conceivable evolutionary scenario is that the ancestor of the Aquificaceae acquired a harp gene, for example, by horizontal gene transfer from an archaeon (Nickel et al. 2017; Schencking et al. 2020). Since HARPs have the basic enzymatic capacity to catalyze the RNase P reaction, although in many cases with relatively low efficiency (Nickel et al. 2017; Schwarz et al. 2019; Li et al. 2022), the ancestor of Aquificaceae may have been able to improve the efficiency of its HARP enzyme with relatively small adaptations. This development then enabled the progenitor strain to abandon the classical RNase P genes (rnpB and rnpA). Potential reasons for this enzyme replacement include constraints for genome size reduction, simplification of gene expression and enzyme assembly, or increased thermo-tolerance. Intrigued by such an evolutionary scenario, we considered the possibility that the presence of two types of RNase P within the same bacterium might have relaxed the constraints for the RNP enzyme to conserve its canonical structure, as some of the workload in tRNA 5′-end maturation may be carried out by the HARP backup activity. To explore this possibility, we have studied the function of RNP RNases P and HARPs in five bacteria that encode both enzyme types, namely Alkalilimnicola ehrlichii, Halorhodospira halophila, Thioalkalivibrio nitratireducens (all γ-proteobacteria), Methylacidiphilum infernorum (Verrucomicrobia, Methylacidiphilae), and Thermodesulfatator indicus (Thermodesulfobacteria) (Nickel et al. 2017). Using Escherichia coli RNase P as reference, we compared the predicted RNase P RNA (P RNA) structures encoded in these “dual RNase P” bacteria, and investigated in vitro processing activities of their HARPs and their P RNAs in RNA-alone and RNP holoenzyme reactions. Furthermore, we investigated the complementation efficiency of P RNAs, RnpA proteins and HARPs from these bacteria in conditionally lethal rnpB (E. coli) and rnpA (Bacillus subtilis) mutant strains. Finally, we scrutinized T. indicus P RNA in more depth, as its rnpB gene encodes an expansion segment in the P12 region of P RNA.

RESULTS

P RNA structures

Predicted P RNA structures are shown in a secondary structure presentation adjusted to the crystal structure of the Thermotoga maritima (Tma) RNase P holoenzyme (Fig. 1; Supplemental Fig. S1). While the γ-proteobacterial P RNAs of A. ehrlichii (Aehr), H. halophila (Hhal), and T. nitratireducens (Tnit) are predicted to adopt canonical type A structures (Supplemental Fig. S1A–C), those of M. infernorum (Minf) and T. indicus (Tind) show notable deviations (Fig. 1A,B). Minf P RNA has an extended P9 helix, and P16.1, P18.1 and P19 expansion elements. P16.1 (or other deviations in the P15/P16/17 region) and P19 are also found in other members of the Planctomycetes-Verrucomicrobia-Chlamydiae bacterial superphylum (PVC group; Herrmann et al. 1996, 2000; Butler and Fuerst 2004). However, the P18.1 element is rarely found in P RNA type A architectures. In contrast to Chlamydiaceae species (Herrmann et al. 2000), Minf P RNA still encodes the L15 5′-GGU motif interacting with the CCA end of tRNA molecules (Fig. 1B; Kirsebom and Svärd 1994). The most conspicuous feature of Tind P RNA is a considerably expanded P12 element (Fig. 1A).

FIGURE 1.

Predicted secondary structures of RNase P RNAs (P RNAs) from (A) T. indicus, (B) M. infernorum, and (C) T. maritima in a secondary structure presentation attuned to (D) the 3D structure of the T. maritima P RNA as part of the RNase P holoenzyme (Massire et al. 1998; Reiter et al. 2010). (A–C) Coaxially stacked helices are of the same color and the five regions with highest sequence conservation (CR-I to CR-V), which cluster in two regions, are highlighted; CR-I and CR-V overlap with helix P4. The gray broken line separates the catalytic (C-) and specificity (S-) domains. Tertiary contacts (according to the T. maritima RNase P holoenzyme structure; Reiter et al. 2010) are marked by thin dotted lines connecting boxed or circled elements; question marks were added to indicate that formation of this contact in the respective RNA is unclear due to structural variation. Sequences that lack P19 possess instead a 4 nt linker (R19) (Massire et al. 1998). Noncanonical structure elements in panel B (P16.1, P18.1) are drawn in gray. The 5′-GGU motif in loop 15 (L15) interacting with the CCA end of tRNA molecules is highlighted in panel B. (D) Adapted with permission from Gößringer et al. (2021). Structural elements are colored as in panel A–C. Ochre spheres represent two active site metal ions (Me1/2) and two structurally important metal ions (Me3, Me4; Reiter et al. 2010).

RNA-alone and holoenzyme activity assays

Processing of a bacterial precursor tRNA^Gly by the five P RNAs alone as well by holoenzymes reconstituted with the RnpA protein of B. subtilis (Bsu) was analyzed using E. coli (Eco) P RNA as reference. P RNA alone reactions were performed exclusively under single-turnover (sto) conditions to focus on catalytic performance, as product release limits RNA-alone reactions at 100 mM Mg²⁺ (Reich et al. 1988; Tallsjö and Kirsebom 1993). For easier handling and reproducibility of the manual kinetic measurements, processing assays were performed at 25°C instead of 37°C to slow down the single-turnover reactions (based on activities obtained with E. coli P RNA at pH 7.4). Recombinant Bsu RnpA, purified as reported (Niranjanakumari et al. 1998), was used because it has proven in our hands to have very robust and reproducible activity. Bacterial RnpA proteins share the same fold despite limited sequence similarity, bind to the conserved catalytic core structure of bacterial P RNAs (Reiter et al. 2010) and were shown in numerous studies to be functionally exchangeable in vitro (Hansen et al. 2001; Buck et al. 2005; Wegscheid et al. 2006) as well as in vivo (Gößringer and Hartmann 2007). Moreover, usage of recombinant E. coli RnpA might have conferred an advantage on E. coli P RNA relative to the other five P RNAs under investigation.

RNase P assays were performed in KN buffer (see Materials and Methods; Wegscheid and Hartmann 2006), a buffer that was optimized for functional analyses of E. coli ribosomes to closely mimic physiological conditions (Dinos et al. 2004). RNA-alone reactions were assayed at 100 mM Mg²⁺, a standard concentration that has been used in numerous previous studies for this reaction. RNase P holoenzyme activity was measured at 4.5 mM Mg²⁺, which is close to the estimated free Mg²⁺ concentration (1–2 mM) in E. coli cells (Alatossava et al. 1985) and where the RNA-alone reaction is negligible in the applied reaction time window of 15 s to 15 min, in line with previous findings (Guerrier-Takada et al. 1986). In the chosen setup, activities ranged from ∼0.1 (Tind) to ∼1.7 min⁻¹ (Aehr) in the RNA-alone reaction and from ∼0.2 (Tind) to ∼3 min⁻¹ (Aehr) in the holoenzyme reaction (Fig. 2A,B; Supplemental Table S1). At the assay temperature of 25°C, highest activities were measured with Aehr P RNA and lowest activities with Tind P RNA. The relatively low activity of Tind P RNA at 25°C did not change substantially when applying different RNA folding protocols (Supplemental Fig. S2; Supplemental Table S2). Taken together, the activity measurements demonstrated that all tested P RNAs are catalytically active, though to different extents. We are aware that the relative activities of the tested P RNAs (Fig. 2) may change when complexed with their native RnpA cofactors and when acting in their respective host environments.

FIGURE 2.

Pre-tRNA processing activity of P RNAs from E. coli (Eco), A. ehrlichii (Aehr), H. halophila (Hhal), M. infernorum (Minf), T. indicus (Tind), and T. nitratireducens (Tnit) under single turnover conditions. (A) RNA-alone reaction containing 25 nM P RNA and <1 nM of 5′-³²P-endlabeled pre-tRNA^Gly were conducted at 25°C in the presence of 100 mM Mg²⁺. (B) For the holoenzyme reaction, 50 nM B. subtilis P protein was added to 10 nM P RNA, and substrate (<1 nM of 5′-³²P-endlabeled pre-tRNA^Gly) processing was analyzed in the presence of 4.5 mM Mg²⁺ at 25°C. The rate constants k_obs (min⁻¹) obtained under these conditions are given for each P RNA in the RNA-alone or holoenzyme reaction, based on three or more independent experiments for each enzyme. Error bars are standard deviations (SD). For more details, see Materials and Methods.

Complementation analysis of heterologous rnpB genes in the RNase P mutant strain E. coli BW

For the analysis of (heteterologous, mutant) P RNA or protein-only RNase P function under in vivo conditions, we consider the E. coli BW strain as the best available test strain that has been used for this purpose in numerous previous investigations (Wegscheid and Hartmann 2006, 2007; Marszalkowski et al. 2008, 2021; Gobert et al. 2010; Li et al. 2011; Walczyk et al. 2016; Gößringer et al. 2017; Nickel et al. 2017). In the E. coli BW strain, the P RNA gene (rnpB) is under control of an arabinose-inducible promoter. In the absence of arabinose and presence of glucose as carbon source, rnpB expression is shut down resulting in growth arrest (Wegscheid and Hartmann 2006). Expression of a functional RNase P RNA gene from a low-copy plasmid can restore growth (for details, see Supplemental Material).

The homologous Eco rnpB gene as well as Aehr, Hhal and Tnit rnpB genes showed successful complementation (Fig. 3A). Effective complementation by Minf and Tind rnpB genes became only evident when the Eco P protein (RnpA) was simultaneously overexpressed and when the heterologous rnpB gene was embedded into the 3′-flanking region of the E. coli rnpB gene (Fig. 3B,C). RnpA overexpression increases the steady-state levels of the RNase P holoenzyme (Wegscheid and Hartmann 2006, 2007; Kim and Lee 2009) and the 3′-flanking region of E. coli rnpB harbors an RNase E cleavage site whose utilization stabilizes P RNA transcripts against degradation (Lundberg and Altman 1995; Kim et al. 2005). Nonetheless, growth of E. coli BW in the presence of glucose remained slower with Tind and particularly Minf rnpB under glucose conditions relative to complementation with the γ-proteobacterial rnpB genes (compare Fig. 3A, columns 2–5, with Fig. 3B, columns 5 and 8, growth for 16 h with glucose).

FIGURE 3.

Functionality of heterologous P RNAs in the RNase P complementation test strain E. coli BW. (A) Analysis of P RNAs (rnpB genes) from A. ehrlichii (Aehr), H. halophila (Hhal), and T. nitratireducens (Tnit) using the homologous E. coli rnpB gene (Eco) as positive control; all P RNAs were expressed from the low copy vector pACYC177 under control of the native E. coli rnpB promoter (for details, see the Supplemental Material). Colony growth was documented after 16 h of incubation at 37°C on agar plates supplemented with arabinose (Ara, permissive conditions) or glucose (Glu, nonpermissive conditions). Three independent experiments gave comparable complementation results. (B) Analysis of in vivo functionality of M. infernorum (Minf) and T. indicus (Tind) rnpB genes in E. coli BW. Complementation efficiency was additionally analyzed by simultaneous plasmid pBR322-borne overexpression of the E. coli RNase P protein (RnpA; indicated by EcoRnpA↑) and/or by fusing the heterologous rnpB gene not only to the E. coli rnpB promoter, but also to the 3′-flanking region of E. coli rnpB (Minf-Eco 3′, Tind-Eco 3′) to attenuate P RNA decay in E. coli. Colony growth in the presence of arabinose or glucose was documented after 16, 28, and 42 h at 37°C. Five independent experiments gave comparable complementation results. (C) The 5′- and 3′-ends of Minf and Tind P RNA as annotated in their genomes are shown at the top; Minf and Tind P RNA transcripts equipped with the 3′-flanking region of E. coli rnpB are illustrated at the bottom. Boxes mark the nucleotides in helix P1 that are identical between the native and engineered P RNAs. The thin vertical arrows at the bottom indicate RNase E cleavages and the thick arrow the mature 3′-end after trimming by exonucleolytic activities (Kim et al. 2005).

Complementation analysis of heterologous rnpA genes in the B. subtilis RNase P mutant strain d7

We previously constructed a bacterial rnpA test strain (B. subtilis d7) to investigate the function of heterologous or mutant RNase P protein cofactors in vivo (Gößringer et al. 2006; Gößringer and Hartmann 2007). We showed successful complementation (growth rescue) of this strain under nonpermissive conditions upon expression of heterologous rnpA genes from a wide variety of bacteria, but not with genes expressing archaeal or eukaryal RNase P protein subunits (Gößringer and Hartmann 2007). Thus, the d7 strain was considered to be suitable for the functional analysis of bacterial RnpA proteins under investigation in the present study. The rnpA gene is under control of a xylose-dependent promoter in B. subtilis strain d7. Cultivation in the presence of glucose leads to depletion of the RNase P holoenzyme and growth arrest that is rescued when d7 cells are transformed with a plasmid expressing a functional RnpA protein (Gößringer et al. 2006; Gößringer and Hartmann 2007). All five heterologous RnpA proteins (sequences aligned in Supplemental Fig. S3) were able to rescue growth in the presence of glucose, but with different efficiencies. d7 cells expressing Tind and Minf RnpA grew moderately slower than the control strain with plasmid-borne expression of Bsu RnpA, while d7 cells complemented with Aehr, Hhal, or Tnit rnpA grew markedly slower. Nevertheless, these findings basically confirm the functionality of these putative RnpA proteins as RNase P subunits (Fig. 4).

FIGURE 4.

Functionality of RNase P proteins (rnpA genes) from A. ehrlichii, H. halophila, M. infernorum, T. indicus, and T. nitratireducens in the RNase P protein complementation test strain B. subtilis d7, including B. subtilis (Bsu) rnpA as positive control. The homologous/heterologous rnpA genes were expressed in B. subtilis d7 from vector pDG148(S/X) under control of the spac promoter. Single colonies of B. subtilis d7 transformed with different rnpA expression plasmids were spotted onto LB agar plates supplemented with xylose (Xyl, permissive conditions) or glucose (Glu, nonpermissive conditions). Complementation efficiency was documented after 19, 24, and 45 h (shown here) of incubation at 37°C. The figure shows one representative experiment out of three independent ones. For further details, see Materials and Methods.

HARP activities

We also tested processing of pre-tRNA^Gly by recombinant HARPs from the same group of bacteria. All HARP proteins, displaying high sequence similarity (Supplemental Fig. S4), were confirmed to be soluble (Supplemental Fig. S5). At a protein concentration of 50 nM, only Tind HARP showed RNase P activity using A. aeolicus HARP (Aq880) as reference (Fig. 5A, lanes 1–8). At a 10-fold higher HARP concentration (500 nM), cleavage was additionally observed for Minf and Hhal HARP (Fig. 5A, lanes 12 and 17). In the case of Hhal HARP, we repeatedly observed that freshly prepared protein was active but lost activity upon storage, for unknown reasons. In the genetic complementation analysis, only Tind HARP and Aq880 were able to rescue growth of E. coli BW under nonpermissive (glucose) conditions, though less effectively than Eco rnpB (Fig. 5B). Although in vivo complementation assays and in vitro cleavage assays, the latter performed with an excess of enzyme over substrate for 1 h, cannot be directly correlated, both assays nonetheless support the notion that the RNase P activities of Aq880 and Tind HARP are more robust than those of the other HARPs investigated here.

FIGURE 5.

HARP activity in vitro and in vivo. (A) Processing of pre-tRNA^Gly by HARPs from H. halophila (Hhal), T. nitratireducens (Tnit), A. ehrlichii (Aehr), M. infernorum (Minf), T. indicus (Tind), and A. aeolicus (Aq880, positive control), under single turnover conditions (<1 nM 5′-³²P-endlabeled pre-tRNA^Gly). In lanes 1–14, the substrate and 50 nM (lanes 1–8) or 500 nM HARP (lanes 9–17) were incubated at 37°C in the presence of 4.5 mM Mg²⁺ for 60 min (120 min in lanes 16 and 17); con. 1, 2: incubation of substrate for 60 min (lane 15) or 120 min (lane 16) without HARP enzyme. Tag-free variants of recombinant HARPs (lanes 2, 4, 10, and 17) were additionally analyzed to assess the influence of His tags (C-His, carboxy-terminal, or N-His, amino-terminal) on processing activities. (B) In vivo complementation ability of the same set of HARP proteins in E. coli BW. Heterologous harp genes were expressed in E. coli BW from vector pDG148(S/X) under control of the spac promoter. The E. coli BW strain containing the empty vector pDG148(S/X)(vector) was included as negative control and cells transformed with plasmid pACYC177 encoding the E. coli rnpB gene (Eco rnpB) as positive control. Single colonies of E. coli BW containing the harp or rnpB expression vector and grown in the presence of arabinose were washed and resuspended in 800 µL LB medium before 10 µL of cell suspension were spotted onto LB agar plates supplemented with arabinose (Ara) or glucose (Glu). After 24 h incubation at 37°C, colony formation was documented. Panels A and B show representative examples out of three independent experiments in each case.

Thermostability of Tind P RNA

As the processing reactions illustrated in Figure 2 were conducted at 25°C, we considered the possibility that Tind P RNA may perform better at temperatures within the natural growth temperature range of the bacterium (55°C–80°C, optimally 70°C; Moussard et al. 2004). We thus compared processing efficiencies by Tind P RNA at 25°C, 50°C, 60°C, and 70°C with those catalyzed by P RNAs from E. coli and from another thermophile, T. maritima (Tma). Corresponding holoenzymes were reconstituted with Tma RnpA (Fig. 6A,B; Supplemental Table S3). In this setup, P RNA alone and holoenzyme activities measured at assay temperatures of 50°C and 60°C were very similar for Tind and Tma P RNAs, but were not significantly higher, or even tended to be lower, than those obtained with the mesophilic Eco P RNA (Fig. 6). We generally observed substantial fluctuation of holoenzyme activities at 50°C and 60°C (Fig. 6). Evidently, the holoenzyme, its subunits and/or the substrate are sensitive to denaturation at these temperatures. This is in line with a previous in vitro investigation of the Tma RNase P holoenzyme, where activity (and experimental error) peaked at 50°C, followed by a sharp activity drop at 55°C to 60°C (Paul et al. 2001). At 70°C, P RNA-alone activities generally dropped (Fig. 6A), yet the holoenzyme containing Tind P RNA performed relatively best among all three, and was the only one that had higher activity at 70°C than at 25°C (Fig. 6B). This can be taken as evidence for the activity of Tind P RNA at the high growth temperatures of its host. However, activity of the Tind holoenzyme was higher at 50°C and 60°C (and similar to those of the Tma holoenzyme) than at 70°C. Very low activity at 70°C may include contributions from partial unfolding of the pre-tRNA transcript lacking any stabilizing nucleoside modifications, from increases in the equilibrium dissociation constants of holoenzyme-substrate and P RNA-RnpA complexes, decreased occupancy of catalytically relevant Mg²⁺ binding sites or denaturation of the Tma RnpA under in vitro conditions. It should be noted here that processing assays presented in Figure 6 were conducted with a full-length transcript of Tind P RNA. We later noticed that the distal part of P12 is excised from the RNA in T. indicus as well as E. coli cells (see below). Thus, the activity assay performed here with the unfragmented Tind P RNA may not fully mirror the RNA's functionality in its fragmented state within the T. indicus host.

FIGURE 6.

Comparison of processing activities of P RNAs from T. indicus (thermophile), E. coli (mesophile), and T. maritima (thermophile) at elevated assay temperatures. (A) RNA-alone single turnover (sto) reactions containing 25 nM P RNA and <1 nM 5′-³²P-endlabeled pre-tRNA^Gly were performed in KN buffer supplemented with 100 mM MgCl₂ at 25°C, 50°C, 60°C, or 70°C. Before reaction start, pre-tRNA and P RNA were preincubated separately for 5 min at 55°C and 5 min at the respective assay temperature. (B) Corresponding RNase P holoenzyme reactions; holoenzymes were reconstituted with the RnpA protein from T. maritima. In the holoenzyme reaction, 25 nM P RNA were assembled with 25 nM T. maritima P protein and analyzed in KN buffer supplemented with 4.5 mM MgCl₂ in the presence of 125 nM pre-tRNA substrate (multiple turnover, mto) at 25°C, 50°C, 60°C, or 70°C. The data shown in panels A and B are based on three independent experiments (±SD) each. For RNA preincubation and holoenzyme assembly, see Materials and Methods.

Levels and quality of heterologous P RNAs in E. coli BW

The heterologous P RNAs expressed in E. coli BW were analyzed by northern blotting using T7 RNA polymerase in vitro transcripts (T7 transcripts) of these P RNAs as length markers. For BW bacteria complemented with Aehr, Hhal, Tnit, and Minf rnpB genes, signals corresponding to the expected full-length P RNAs were detected (Fig. 7). In the case of Minf rnpB, overexpression of the E. coli RnpA protein increased the levels of Minf P RNA in the E. coli BW strain (Fig. 7, lanes 12 and 13). For Tind P RNA, we initially failed to see a signal in northern blots using a probe directed against the P12 region (complementary to nt 139–192 in Fig. 1A). Then, with a new probe specific for the 5′-portion of the RNA, almost no full-length Tind P RNA was detected, but instead a smaller fragment of ∼135 nt accumulated in BW bacteria expressing Tind rnpB (Fig. 8A, lane 3). With a probe specific for the 3′-portion of Tind P RNA, a fragment of ∼200 nt was detected (Fig. 8A, lane 8). These findings indicated cleavage of Tind P RNA on both sides of the A/U-rich part of helix P12 (around nt 135 and 205) in E. coli cells, resulting in excision of the distal part of P12 (∼nt 135–205). Overexpression of E. coli RnpA somewhat increased the level of full-length Tind P RNA (Fig. 8A, compare lanes 4 and 9 with 3 and 8). This effect was also evident when Tind RnpA was overexpressed in E. coli BW (Fig. 8A, lane 5), thus further confirming that T. indicus expresses a functional RnpA protein. To address whether Tind P RNA cleavage in the P12 region also takes place in the natural host, we analyzed total RNA from T. indicus cells by northern blotting. This revealed the same kind of fragmentation and truncation of Tind P RNA in the native host (Fig. 8B, compare lanes 2 and 5 with 3 and 6). 5′/3′-RACE using total RNA from T. indicus cells identified a major cluster of 5′- and 3′-ends in the A/U-rich region centering around nt 135 and 205 (Table 1; Supplemental Fig. S6), in line with the estimated lengths of the northern blot signals (Fig. 8A, lanes 3–5, 8 and 9). We then replaced the expanded P12 element of Tind P RNA with the short P12 element of Tma P RNA to examine whether this prevents fragmentation of the RNA in the E. coli BW host. The chimeric P RNA termed “Tind-P₁₂-Tma” was 87 nt shorter than the primary transcript of Tind P RNA (Fig. 9). In the northern blot, only a signal corresponding to the full-length RNA was now detected in E. coli BW (Fig. 8A, lanes 10 and 11). Complementation of E. coli BW cells with the Tind-P₁₂-Tma rnpB gene was most efficient when E. coli RnpA was overexpressed and when this gene carried the 3′-flanking region of E. coli rnpB (Eco 3′; Fig. 10, columns 5–8, row 4, growth after 16 h in the presence of glucose). Surprisingly, complementation with Tind-P₁₂-Tma or Tind-P₁₂-Tma-Eco 3′ rnpB was less efficient than with Tind or Tind-Eco 3′ rnpB (Fig. 10, compare columns 2 and 3 with 5 and 6, glucose conditions). One possibility is that cleavage of Tind P RNA in the P12 region favors folding of the RNA into its catalytically active structure.

FIGURE 7.

Northern blot analysis of A. ehrlichii (Aehr), H. halophila (Hhal), T. nitratireducens (Tnit), and M. infernorum (Minf) P RNA (rnpB) expression in E. coli BW. Minf-Eco 3′ rnpB, Minf P RNA embedded into the 3′-flanking region of E. coli rnpB. Total RNAs were isolated in exponential growth phase; the DIG-labeled hybridization probes are specified in Supplemental Table S6. In lane 13, the E. coli RnpA protein was simultaneously overexpressed (indicated by EcoRnpA↑). Lanes “T7 ivt”: T7 in vitro transcripts of the respective P RNAs (marked by arrows) that were used as positive and length controls (for the exact 5′- and 3′-ends of the T7 transcripts, see Supplemental Table S4); lanes “vec.”: total RNA extracted from E. coli BW transformed with the empty vector pACYC177. Twin bands for Hhal and Tnit P RNAs expressed in E. coli are likely variants with minor length variations at the 3′-end. Three independent experiments gave identical results. 5S rRNA was used as loading control. For more details, see Materials and Methods.

FIGURE 8.

Northern blot analysis of T. indicus (Tind) P RNA expressed in E. coli BW or T. indicus. Total RNAs were isolated in exponential growth phase. DIG-labeled probes specific for the 5′- or 3′-region of Tind P RNA were complementary to nt 1–119 (5′-probe) and nt 312–339 (3′-probe) (see Fig. 1A; Supplemental Table S6). (A) Lanes “T7 ivt”: T7 in vitro transcript (see Supplemental Table S4) of Tind P RNA used as positive and length control; lanes “vec.”: total RNA extracted from E. coli BW transformed with the empty vector pACYC177. Simultaneous plasmid pBR322-borne overexpression of the E. coli or T. indicus RnpA protein in BW bacteria (indicated by EcoRnpA↑ or TindRnpA↑) is indicated above the lanes. The Tind P RNA variant Tind-P₁₂-Tma- Eco 3′ (see Fig. 9) was expressed in lanes 10 and 11. The position and length (in kb) of bands of an RNA size marker (Low Range ssRNA Ladder, NEB) loaded onto the gels is indicated at the right margin of each blot. 5S rRNA was used as loading control. (B) Comparison of northern blot signals for Tind P RNA expressed in E. coli BW (lanes “Tind-Eco 3′ rnpB BW”) or in T. indicus cells (lanes “Tind total RNA”). For more details, see Materials and Methods. The northern blots shown in panels A and B are in each case representative examples out of three independent experiments. The signals corresponding to the in vivo-generated 5′-fragment(s) (5′F) and 3′-fragment(s) (3′F) of Tind P RNA are marked by asterisks and red lettering.

TABLE 1.

3′- and 5′-ends of native T. indicus P RNA and fragments thereof, mapped by RACE

FIGURE 9.

Illustration of the P12 region in the native Tind P RNA primary transcript (left) and in the hybrid P RNA Tind-P₁₂-Tma carrying the short P12 element of T. maritima P RNA (P12 Tma; in magenta).

FIGURE 10.

Functionality of heterologous P RNAs in the RNase P complementation test strain E. coli BW. For details, see legends to Figures 3 and 9. Column 1: total RNA extracted from E. coli BW transformed with the empty pACYC177 plasmid (vector) and pBR322-based E. coli RnpA expression plasmid (EcoRnpA↑); column 2: Tind, expression of T. indicus P RNA with 5′- and 3′-ends as annotated in the T. indicus genome, but under control of the E. coli rnpB promoter; column 3: Tind-Eco 3′, as column 2, but Tind P RNA embedded into the 3′-flanking region of E. coli rnpB; column 4: as column 3, but with simultaneous E. coli RnpA overexpression; columns 5–8: corresponding variants of P RNA Tind-P₁₂-Tma. Two independent experiments with two clones each gave comparable results.

Active transcription of rnpA and harp genes in T. indicus

We finally confirmed by qRT-PCR that harp and rnpA genes are actively transcribed in T. indicus (Fig. 11). The levels of rnpA and harp mRNAs are 10- and 14-fold lower, respectively, than 5S rRNA levels. We are aware that this does not yet inform on the level of HARP and RnpA proteins in T. indicus (see also Discussion).

FIGURE 11.

Quantification of rnpA mRNA, harp mRNA and rrf (5S rRNA) RNA levels in total RNA prepared from T. indicus (DSM_15286) cells grown to mid-exponential phase. After reverse transcription, 2.5 µL of 1:20 and 1:80 dilutions (in ddH₂O) of the cDNA sample were subjected to qPCR using rnpA-, harp- or rrf-specific primer pairs (Supplemental Table S8) as detailed in Materials and Methods. (A) C_T values of rnpA mRNA, harp mRNA and 5S rRNA in T. indicus determined using two different cDNA sample dilutions. (B) Relative levels of rnpA and harp mRNAs normalized to 5S rRNA levels (2^−ΔC_T; ΔC_T = C_{T(rrf, rnpA or harp)} − C_T(rrf)). (C) Data table for panels A and B. C_T values (±SD) were based on six independent experiments each.

DISCUSSION

All bacteria with “dual RNase P” activities analyzed here express functional RNP RNases P. Regarding rnpB complementation in E. coli BW bacteria, we observed that expression of Minf P RNA impaired growth of the BW strain under permissive conditions in the presence of arabinose as carbon source (Fig. 3B, columns 3 and 4 vs. 1, arabinose conditions). This was not the case for the four other bacteria investigated here (Fig. 3A,B). How can the retarded growth of BW bacteria expressing Minf P RNA under permissive conditions be explained? Under permissive conditions, the heterologous P RNA competes with the native Eco P RNA for binding to the limited amounts of Eco RnpA, such that only a fraction of both P RNAs capture a protein cofactor. This could result in a growth defect if the hybrid holoenzyme functions less efficiently and/or if the Minf P RNA is rapidly turned over in E. coli. Overexpression of Eco RnpA (partially) rescued growth of the Minf rnpB complementation strain under arabinose and glucose conditions (Fig. 3B, compare columns 4 and 5). Rescue in the presence of arabinose can be explained by formation of more native holoenzyme (Kim et al. 2005) to support tRNA 5′-end maturation. Partial growth rescue in the presence of glucose can be attributed to the assembly of more hybrid holoenzymes and the resulting increase in cellular RNase P activity. This notion is consistent with higher levels of Minf P RNA in E. coli BW upon overexpression of E. coli RnpA (Fig. 7, lanes 12 and 13).

Noteworthy, only the HARP of T. indicus showed in vitro RNase P activity comparable to that of the type HARP Aq880 and was able to compensate the RNase P deficiency of E. coli BW cells grown under nonpermissive conditions. This is surprising in light of the marked sequence conservation of HARPs (Supplemental Fig. S4). Possible explanations may be related to the oligomerization behavior of HARPs (Feyh et al. 2021). However, the inactive HARPs also form oligomers (Supplemental Fig. S7), making a simple correlation of activity and oligomerization state unlikely. We showed for the A. aeolicus enzyme (Aq880) by mass photometry that the recombinant protein exists as an ensemble of different oligomers from dimers to dodecamers, suggesting dynamic oligomerization; in comparison, Hhal HARP formed predominantly dodecamers (Feyh et al. 2021). The size exclusion chromatography (SEC) profiles in Supplemental Figure S7 show that smaller oligomers than dodecamers prevail in the case of the recombinant Minf and Tind HARPs, while dodecamers dominate in the case of Aehr and Tnit HARPs. Unpublished mass photometry data indicate that the Minf and Tind HARPs tend to form mainly dimers and tetramers, although we also observed variation in the oligomerization behavior. Such variation might be affected by protein concentration in some cases. However, the superimposed SEC profiles of Hhal_HARP/Hhal_HARP_nHis and Tnit_HARP/Tnit_HARP_nHis, respectively (Supplemental Fig. S7), do not confirm this supposition: the His-tagged protein variants were loaded onto the column at lower concentrations (see Supplemental Methods, paragraph “Size exclusion chromatography”) than the untagged variants without significant changes of the elution profiles. In conclusion, a coherent picture how oligomeric state and activity are correlated is still lacking.

The inactivity or low and volatile activity of Hhal, Aehr, Minf, and Tnit HARPs might also be explained by the sensitivity of HARP oligomerization and active conformation to conditions such as temperature, pH, salinity, stress factors or metabolism. This would be in line with the fact that the HARP-expressing bacteria analyzed here are adapted to more extreme environmental conditions and utilize specialized metabolic pathways (see beginning of Supplemental Methods). Our in vitro conditions for the measurement of HARP activities or the intra- and extracellular milieu of E. coli cultures might have prevented the HARPs found to be inactive in our study from adopting a catalytically active oligomerization state or conformation. Oligomerization may be determined by the less conserved regions of HARPs, such as the carboxy-terminal region. Indeed, deletion of the carboxyl terminus comprising the terminal α-helix of Aq880 abolished activity and prevented formation of tetramers and larger oligomers (Feyh et al. 2021). There might also be cofactors of HARPs in these extremophilic bacteria that favor or induce a certain oligomeric state or conformation of their HARPs that is required for RNase P activity. Such cofactors might only be present under certain conditions, particularly if the RNase P function of HARPs was not the primary function in these bacteria, similar to the situation in archaea where HARP gene knockouts did not compromise tRNA 5′-end maturation under a variety of growth conditions (Schwarz et al. 2019).

Among the analyzed bacteria, T. indicus is phylogenetically most closely related to the Aquificaceae (Zhu et al. 2019), which correlates with their common habitats (deep-sea hydrothermal vents), their chemolithoautotrophic metabolism and similarities in lipid/fatty acid composition (Moussard et al. 2004). Overall, our findings suggest that Tind HARP has the potential to contribute to tRNA 5′-end maturation in T. indicus. This might relax the evolutionary constraints for the Tind RNP RNase P, as the essential RNase P function is shouldered by two separate activities, which may entail an increasing tolerance toward genetic changes pertaining to the rnpA and rnpB genes. This situation may resemble the predicted prestage in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes. One idiosyncrasy of the Tind P RNA is the P12 expansion segment that is excised in vivo, resulting in a shortened, fragmented P RNA. Although speculative at present, the development of this peculiarity might have been facilitated in the context of redundant RNase P activities. However, this P RNA expansion is the opposite of what one may expect in an evolutionary RNP to protein-only transition, namely that structural P RNA elements rather disappear, possibly also favored by constraints to reduce genome size as suggested in other hyperthermophiles such as the Aquificaceae with genomes condensed to ∼1.6 Mbp (Lechner et al. 2014). T. indicus has a quite compact genome (∼2.3 Mbp, Anderson et al. 2012), but not as compact as Aquificaceae genomes.

It should be noted here that the in vitro experiments were conducted with unfragmented Tind P RNA including the entire P12 expansion segment. Attempts to anneal the 5′- and 3′-segments of fragmented Tind P RNA in vitro for activity testing of P RNA after P12 truncation failed. It thus remains an open question whether the fragmented Tind P RNA would be more active than the primary transcript. Higher activity of the fragmented Tind P RNA in vivo would be consistent with our finding that replacement of the native P12 element with the short P12 helix of Tma P RNA abolished fragmentation of Tind P RNA, but simultaneously impaired complementation efficiency in E. coli BW cells (Figs. 8A, 10). This raises the possibility that extension of P12 in Tind P RNA and its intracellular fragmentation might play a role in folding of the RNA into its active conformation in the cellular context (see below) or may affect intracellular enzyme localization.

The failure to anneal the 5′- and 3′-segments of fragmented Tind P RNA to form a functional ribozyme is in contrast to a previous study by Guerrier-Takada and Altman (1992). They reconstituted ribozyme activity from combining independently transcribed 5′- and 3′-halves of E. coli P RNA (separated in the P12 region). Ribozyme activity could also be restored by fragment combination in the case of the thermostable P RNA from T. thermophilus (Schlegl et al. 1994). Another remarkable example is the assembly of mitochondrial ribosomes from 12 discontinuously transcribed rRNA fragments in the green alga Chlamydamonas reinhardtii (Boer and Gray 1988). Against this background, our failure to reconstitute Tind P RNA activity by combining 5′- and 3′-halves remains unclear. Possible reasons might be, in this particular case, aberrant intramolecular folding of the Tind P RNA 5′- and 3′-halves or aberrant annealing of the two halves. In-depth exploration of different annealing protocols combined with fine-tuning of RNA fragment ends might yet eventually reveal conditions for successful reconstitution. However, it should be noted in this context that Tind P RNA is encoded in vivo by a single continuous rnpB gene and its fragmentation occurs post-transcriptionally, thus differing from the assembly of mitochondrial ribosomes from discontinuously transcribed rRNA fragments. Accordingly, the capacity to assemble a functional P RNA from separately transcribed 5′- and 3′-halves appears biologically irrelevant for T. indicus cells.

The P6 pseudoknot of Tind P RNA differs from that in the majority of type A RNase P RNAs. P6 formation was shown to be critical for catalytic activity (Mao et al. 2018). We resequenced a PCR fragment of the rnpB gene derived from T. indicus genomic DNA and could confirm the sequence in the published genome (NCBI Reference Sequence: NC_015681.1). Commonly, at least one unpaired nucleotide (frequently a U or C; Haas and Brown 1998) separates the 5′-strand of helix P17 and the 3′-strand of helix P6. This spacer nucleotide is lacking in Tind P RNA (compare Fig. 1A with Fig. 1B,C), which might hamper formation of the P6 interaction at low temperature owing to reduced conformational flexibility. It can thus not be excluded that this structural peculiarity had contributed to the relatively low activity of Tind P RNA at the low assay temperature of 25°C (Fig. 2).

Tind P RNA is a thermostable RNA, illustrated by an increased proportion of G:C base pairs in helices (relative to P RNAs from mesophiles) and measurable holoenzyme activity at 70°C (Fig. 6B). Surprisingly, the RNA was still able to fold into its active structure in the E. coli host at 37°C (Fig. 3B). This is in contrast to P RNA from another thermophile, Thermus thermophilus (Tth), that was shown to require a preincubation step of ∼55°C in vitro to resolve a severe folding trap and was unable to complement the E. coli BW strain (Marszalkowski et al. 2008, 2021). This raises the possibility that extension of P12 in Tind P RNA and its subsequent fragmentation might lower the activation energy barrier for folding of the RNA into its active conformation, in line with the aforementioned observation that a Tind P RNA variant with the Tma P12 element abolished RNA fragmentation but impaired complementation efficiency in E. coli BW cells. We are aware that the folding trap of Tth P RNA, and its marked attenuation in Tind P RNA, may be of low functional relevance at the optimal growth temperatures (∼70°C) of the two bacteria, but the difference is of mechanistic interest. Noteworthy, the RNAfold prediction for Tth and Tind P RNA reveals differences at the junction between C- and S-domain. Folding studies of mesophilic P RNAs from E. coli and B. subtilis already revealed that formation of P6 and P7 (near the domain junction) is among the late events in the folding pathway (Zarrinkar et al. 1996; Kent et al. 2000). For Tind P RNA, RNAfold predicts a helix (named here “pseudo P11”; Supplemental Fig. S8) that is different from the authentic P11 helix but juxtaposes the 5′- and 3′-strand regions that form P11. If this pseudo P11 indeed forms, it might alleviate formation of the authentic P11 helix by holding its 5′- and 3′-strand regions in proximity to each other, thereby reducing the high activation energy barrier for surmounting the folding trap associated with the predicted aberrant pairing of elements 5′-P6, 5′-P7, 3′-P7, 3′-P10, and 3′-P11 in both P RNAs (Supplemental Fig. S8). As mentioned above, the cotranscriptional P12 expansion segment and its endonucleolytic excision may help in mitigating or avoiding the folding trap.

Tind P RNA is cleaved in the P12 expansion segment in T. indicus as well as in the heterologous mesophilic host E. coli (Fig. 8B), suggesting that ubiquitous RNase(s) are responsible for this activity despite the phylogenetic distance of T. indicus and E. coli. In E. coli, RNase E is known to cleave the P RNA 3′-precursor transcript at two consecutive positions, followed by exonucleolytic removal of one or two 3′-terminal nucleotides (Fig. 3C; Lundberg and Altman 1995; Kim et al. 2005). RNase E cleavage occurs at two positions within the sequence 5′-ACCUG⇓A⇓UUUA that shows some similarity to the 5′-site (sequence 5′-₁₂₇ACCCCAUUUU₁₃₅) of the P12 cleavage region in Tind P RNA (Fig. 9, left structure). As Tind P RNA was expressed in E. coli with the 3′-flanking sequence of the E. coli rnpB gene that contains the RNase E cleavage site, it is a possibility that E. coli RNase E acting on the 3′-precursor of Tind P RNA is concomitantly directed to the internal RNase E site in the P12 expansion segment. The possibility that Tind P RNA is cleaved in the P12 region by RNase E (or possibly by RNase III) in the E. coli host might be tested in the future using corresponding RNase mutant strains of E. coli (Stead et al. 2010). The closest relative of T. indicus with complete genome assembly in the family Thermodesulfobacteriaceae is Thermodesulfobacterium geofontis. It lacks the long P12 expansion present in Tind P RNA, but has a stretch of five U residues in the loop region of P12 (Supplemental Fig. S1D). Considering that the cleavage region in the Tind P12 element also contains a stretch of four consecutive U residues (nt 132-135; Fig. 1A; Supplemental Fig. S6) at the distal end of the lower P12 stem region (Fig. 9), it is a possibility that T. geofontis P RNA might also be cleaved in the P12 region.

In summary, all five bacteria with “dual RNase P” activities were found to encode functional rnpA and rnpB genes. However, only the HARP of T. indicus showed substantial RNase P activity in vitro and in vivo. Bacterial RnpA proteins have generally relatively low expression levels, commonly due to noncanonical start codons (GUG, UUG), suboptimal internal codon usage in their reading frames and low mRNA abundance (Hansen et al. 1985; Looman and van Knippenberg 1986; Panagiotidis et al. 1992; O'Donnell and Jannsen 2001; Feltens et al. 2003). In the majority of bacteria, rnpA is the second gene in a cotranscript with rpmH encoding the ribosomal protein L34 (Hartmann and Hartmann 2003). Recent proteomics indicate that the number of L34 proteins is ∼24,000 and that of RnpA proteins ∼180 per µm³ of E. coli cell volume (∼130-fold higher concentration of L34 relative to RnpA), averaged over various culture conditions with doubling times between 50 and 170 min (Mori et al. 2021). Reference to “µm³ cell volume” is preferable to “per cell” as the average cell volume of E. coli cells may commonly vary from ∼1 µm³ (doubling time ∼150 min, poor carbon source) to ∼2.5 µm³ (doubling time ∼60 min, glucose medium) (Basan et al. 2015). The number of ribosomes per E. coli cell was independently calculated to be 44,000 at a doubling time of 30 min and 8000 at a doubling time of 100 min (Bremer and Dennis 2008), thus being roughly in the same order of magnitude as ∼24,000 L34 proteins per µm³ of cell volume (assuming one L34 protein per ribosome). These findings substantiate the classification of RnpA as a low abundance protein in bacteria. The level of harp mRNA in T. indicus was found to be somewhat lower than those of rnpA mRNA (Fig. 11), but this does not mean that the intracellular level of HARP protein monomers is as low as that of RnpA proteins. The reading frames for the five analyzed HARPs start with AUG codons except for that of M. infernorum (GUG start). At least for A. ehrlichii, H. halophila and T. nitratireducens we see a clear trend toward less tandem rare codons for harp versus rnpA mRNAs based on E. coli codon usage (https://www.genscript.com/tools/rare-codon-analysis). In conclusion, the cellular levels of HARP mono- and oligomers require further investigation on the protein level. Altogether, our findings are consistent with the RNP enzyme being the main tRNA 5′-end maturation activity in four of the five analyzed bacteria. Such an assessment is less evident for T. indicus whose HARP shows robust RNase P activity. In any case, T. indicus provides an example that resembles the predicted prestage in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes, in that a HARP with substantial RNase P activity coexists with RNP RNase P and thus appears to have the capacity to take over the essential RNase P function after minor evolutionary adaptations.

MATERIALS AND METHODS

Genomes and gene loci

For rnpA, rnpB and harp gene sequences and respective genomes, see the Supplemental Material.

Enzymes and kits

DNA polymerases, restriction enzymes and other DNA-modifying enzymes were purchased from Thermo Fisher Scientific or New England Biolabs (NEB) and used as recommended by the manufacturer. The T7 RNA polymerase for in vitro transcription was self-prepared (Gößringer et al. 2014). DNA fragments were extracted from agarose gels and purified using the Wizard SV Gel and PCR Clean-Up System Kit (Promega) or the QIAEX II Kit (Qiagen). Plasmids were prepared from bacterial cell lysates with the Qiagen Plasmid Purification Midi or Maxi Kit (Qiagen) or GeneJET Plasmid-Miniprep-Kit (Thermo Fisher Scientific).

Strains and growth conditions

Escherichia coli DH5α was used for plasmid cloning and preparation, E. coli Rosetta (DE3) and E. coli BL21 (DE3) for protein expression and purification, E. coli BW (Wegscheid and Hartmann 2006) and B. subtilis d7 (Gößringer et al. 2006) were used for in vivo complementation studies of RNase P activity. For details, see the Supplemental Material. Cells on agar plates were incubated at 37°C in a static incubator and cells in liquid bacterial cultures were shaken at 180–220 rpm in a waterbath (Aquatron) or air shaker (GFL). T. indicus (DSM_15286) cells grown to mid-exponential phase were obtained from Dr. Harald Huber, University of Regensburg, Department of Microbiology.

Chromosomal DNA preparation

The protocol used was based on that by Wilson (2001). For details, see the Supplemental Material.

Construction of expression plasmids

Genes (rnpA, rnpB, and those coding for HARPs) were amplified by PCR from genomic DNA or plasmid DNA (see Supplemental Tables S4, S5 for primer sequences). The DSMZ (German Collection of Microorganism and Cell Cultures GmbH) supplied the genomic DNA of Alkalilimnicola ehrlichii (DSM_17681), Halorhodospira halophila (DSM_244), Thermodesulfatator indicus (DSM_15286), and Thioalkalivibrio nitratireducens (DSM_14787). The gene sequences for Methylacidiphilum infernorum HARP (WP_012462349.1) and RNase P (rnpA and rnpB) were commercially synthesized (Biomatik Corporation, Canada). PCR fragments were gel-purified and inserted into vectors via restriction enzyme cloning or Gibson assembly (see Supplemental Tables S4, S5 for primer sequences). All vector constructs were verified by sequencing (Eurofins or Microsynth). For more details, see the Supplemental Material.

Complementation analyses and T7 in vitro transcription

See the Supplemental Material.

Total RNA preparation

Precultures of 3 mL LB medium supplemented with 10 mM arabinose were inoculated with single colonies of E. coli BW strains containing expression vectors for bacterial P RNA. Cells were grown for 8 h at 37°C/200 rpm. Then, 30 mL LB medium supplemented with 10 mM arabinose were inoculated with a 1/2000 volume of the precultures. After 14 h of growth at 37°C/200 rpm, cells were harvested by centrifugation for 7 min at 5000g and washed twice with 25 mL prewarmed LB medium, respectively. The washed cells were resuspended in 10 mL prewarmed LB medium and used to inoculate 50 mL LB medium supplemented with 10 mM glucose to a starting OD₆₀₀ = 0.5. Cells were grown for another 4 h at 37°C/180 rpm before cells were pelleted in 10 OD₆₀₀ aliquots that were shock-frozen in liquid nitrogen for storage at −80°C. Bacterial total RNA was basically prepared as described (Damm et al. 2015; Method I). For details, see method “Total RNA preparation by phenol extraction” in the Supplemental Material.

Northern blot analysis

Northern blot analyses were performed basically as described (Gößringer et al. 2017). For more details, see method “Northern blot analysis” in the Supplemental Material. The digoxigenin-labeled probes were designed to be directed against unique sequences in the different P RNAs on the basis of sequence alignments. The specific probes against 5S rRNA from E. coli and P RNAs from A. ehrlichii, H. halophila, M. infernorum, T. indicus, and T. nitratireducens were synthesized as antisense transcripts by T7 transcription in the presence of digoxigenin-11-UTP (Roche Diagnostics). Except for T. indicus, templates for T7 transcription were prepared by annealing single-stranded DNA oligonucleotides containing a reverse complementary T7 promoter sequence at the 3′-end to a shorter DNA oligonucleotide encoding the T7 promoter sequence. For T7 transcription of the specific probes against E. coli 5S rRNA and T. indicus P RNA (covering the 5′-region up to P12), gel-purified PCR fragments derived from chromosomal DNA of E. coli BW and plasmid DNA of pACYC_Tind-Eco 3′ rnpB were used, respectively. For the detection of the 3′-region of T. indicus P RNA, a synthetic DNA probe with digoxigenin attached to its 5′- and 3′-ends (biomers.net) was used. For sequences of primers and other DNA oligonucleotides, see Supplemental Table S6.

Recombinant HARP enzymes

For the construction of plasmids, expression and purification of recombinant HARPs, see the Supplemental Material.

Bacterial P protein purification

RnpA proteins of B. subtilis and T. maritima were prepared as described (Schencking et al. 2021).

Activity assays

The T. thermophilus pre-tRNA^Gly was used as substrate to analyze 5′-processing by HARP variants, P RNAs or reconstituted RNase P holoenzymes. RNase P holoenzymes were assembled with His tag-purified RnpA proteins from B. subtilis or T. maritima. The 5′-processing activity of P RNAs alone from A. ehrlichii, H. halophila, M. infernorum, T. indicus, and T. nitratireducens (each 25 nM) was analyzed under single turnover conditions (<1 nM of 5′-³²P-endlabeled pre-tRNA^Gly) in KN buffer (20 mM HEPES-KOH, pH 7.4, 150 mM NH₄OAc, 2 mM spermidin, 50 µM spermin, 4 mM ß-mercaptoethanol) supplemented with 100 mM MgCl₂ at 25°C. Pre-tRNA and P RNA were preincubated separately for 5 min at 55°C followed by 20 min at 25°C before combining substrate and enzyme solutions to start the reaction. Holoenzyme assays were performed at 10 nM P RNA, 50 nM B. subtilis RnpA and trace amounts of pre-tRNA substrate (<1 nM of 5′-³²P-endlabeled pre-tRNA^Gly) in KN buffer supplemented with 4.5 mM MgCl₂. Before starting the reaction, pre-tRNA and P RNA were incubated separately for 5 min at 55°C and 20 min at 25°C in KN buffer with 4.5 mM MgCl₂. The RnpA protein was added to the P RNA after the first 15 min of preincubation at 25°C. Then substrate and enzyme solutions were mixed to start the single turnover reactions at 25°C. The activity of in vitro-transcribed T. indicus P RNA was investigated at different temperatures in RNA alone and holoenzyme reactions. Holoenzymes were reconstituted with the RnpA protein of T. maritima. In the RNA-alone reaction, the activity of T. indicus P RNA (25 nM) was analyzed in KN buffer supplemented with 100 mM MgCl₂ under single turnover conditions at 25°C, 50°C, 60°C, and 70°C. Beforehand, pre-tRNA and P RNA were preincubated separately in the same buffer for 5 min at 55°C and 5 min at the respective reaction temperature. Activities of holoenzymes reconstituted from 25 nM P RNA and 25 nM T. maritima RnpA protein were analyzed in KN buffer supplemented with 4.5 mM MgCl₂ in the presence of 125 nM pre-tRNA^Gly substrate (containing trace amounts of 5′-³²P-end-labeled pre-tRNA^Gly) at 25°C, 50°C, 60°C, and 70°C. Before mixing substrate and enzyme solutions, pre-tRNA and P RNA were preincubated separately for 5 min at 65°C, 2 min at 50°C, 2 min at 40°C, 15 min at 25°C, and 2 min at the respective reaction temperature. After 5 min of preincubation at 25°C, the T. maritima RnpA protein was added. Different preincubation conditions were tested for T. indicus P RNA under single turnover conditions (Supplemental Fig. S2). The pre-tRNA processing activity of the different HARP variants was analyzed as described in Nickel et al. (2017).

qRT-PCR analysis

Total RNA (25 µg) was treated with DNase I (NEB; final concentration 0.4 U/µL) in a 50 µL reaction in the presence of 50 U RiboLock RNase Inhibitor (Thermo Fisher Scientific). After incubation for 50 min at 37°C, another 10 U of DNase I were added and the reaction was incubated for another 30 min at 37°C. Thereafter, RNA was extracted and concentrated by acidic phenol-chloroform extraction and ethanol precipitation, respectively. Subsequently, 3 µg of DNA-free total RNA were incubated in the presence of 5 µM Random Hexamer Primers (Thermo Fisher Scientific) for 5 min at 65°C in a total volume of 37.8 µL. Then, the sample was adjusted to 1× reaction buffer (supplied by the manufacturer for SuperScript III reverse transcriptase), 520 µM each dNTP, 5 mM DTT, 120 U RiboLock RNase Inhibitor (Thermo Fisher Scientific) in a total volume of 57 µL and split into three 19 µL aliquots. After 2 min preincubation at 42°C, 1 µL SuperScript III (Thermo Fisher Scientific Invitrogen; 200 U/µL) or 1 µL ddH₂O (minus RT control) was added, followed by incubation for 10 min at 42°C, 30 min at 50°C, 20 min at 55°C, and 15 min at 70°C. Finally, residual RNA was degraded by incubation for 20 min at 37°C in the presence of 1 U RNase H. For quantification, 2.5 µL of 1:20 and 1:80 dilutions (in ddH₂O) of the cDNA sample were subjected to PCR (QuantStudio Real-Time PCR System, Thermo Fisher Scientific) in a total volume of 8 µL containing 1× PowerUp SYBR Green Master Mix (Applied Biosystems) supplemented with gene-specific primer pairs (each 250 nM). Melting curves were recorded at the end of PCR reactions to confirm specificity (inferred from a single peak in the first derivative of the melting curve).

5′- and 3′-RACE

5′- and 3′-RACE were modified based on published protocols (Willkomm et al. 2005; Beckmann et al. 2011). For details, see the Supplemental Material.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.