In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA

Abstract

For catalysis by bacterial type B RNase P, the importance of a specific interaction with p(recursor)tRNA 3'-CCA termini is yet unclear. We show that mutation of one of the two G residues assumed to interact with 3'-CCA in type B RNase P RNAs inhibits cell growth, but cell viability is at least partially restored at increased RNase P levels due to RNase P protein overexpression. The in vivo defects of the mutant enzymes correlated with an enzyme defect at low Mg(2+) in vitro. For Bacillus subtilis RNase P, an isosteric C259-G(74) bp fully and a C258-G(75) bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base pairing to tRNA 3'-CCA but also emphasizing the importance of the base identity of the 5'-proximal G residue (G258). We infer the defect of the mutant enzymes to primarily lie in the recruitment of catalytically relevant Mg(2+), with a possible contribution from altered RNA folding. Although with reduced efficiency, B. subtilis RNase P is able to cleave CCA-less ptRNAs in vitro and in vivo. We conclude that the observed in vivo defects upon disruption of the CCA interaction are either due to a global deceleration in ptRNA maturation or severe inhibition of 5'-maturation for a ptRNA subset.

Figure 1.

Secondary structure illustrations of (A) Bacillus subtilis (type B), (B) Staphylococcus aureus (type B) and (C) Escherichia coli (type A) RNase P RNA according to (7). The two G residues in L15, known (E. coli) or suspected (B. subtilis, S. aureus) to be involved in the interaction with tRNA 3′-CCA, are highlighted by gray ovals. (D) Proposed interaction of a canonical bacterial ptRNA (Thermus thermophilus ptRNA^Gly) with the L15 loop of B. subtilis RNase P RNA. Highlighted nucleotides mark the sites of mutation investigated in this study. The arrow indicates the canonical RNase P cleavage site (between nucleotide –1 and +1).

Figure 2.

Growth curves of SSB318 cells complemented with pHY300 derivatives, carrying S. aureus rnpBwt (squares) or rnpBC238 (triangles with apex at the top) or rnpBC239 (triangles with apex at the bottom) mutant alleles in the presence (+) or absence (−) of IPTG. The better growth of SSB318 bacteria expressing S. aureus rnpBwt (squares) relative to SSB318 carrying the empty vector and grown in the presence of IPTG (open circles) can be explained by the finding that IPTG-induced expression of the chromosomal rnpB gene in the SSB318 mutant strain is weaker than rnpB expression from the native promoter in the original strain W168 used to construct SSB318 ((17), Figure 3 therein). Improved growth was also observed when we expressed B. subtilis rnpBwt from pHY300 in strain SSB318 (data not shown), showing that this effect is not specific for S. aureus rnpB. We conclude that plasmid-borne expression of S. aureus or B. subtilis rnpBwt saturates the cellular RNase P levels in SSB318 bacteria and thereby restores wild-type-like growth.

Figure 3.

Radioactive reverse transcription PCR (RT-PCR) analysis of strain SSB318 complemented with S. aureus rnpBwt or rnpBC238/C239. PCR products were analyzed on a 10% polyacrylamide/8 M urea gel. Lanes 1–30: total RNA from SSB318 complemented with S. aureus rnpBwt (lanes 1–4 and 13-16), rnpBC238 (lanes 5–8, 17–20 and 25–30) or rnpBC239 (lanes 9–12 and 21–24) grown at 37°C in the absence of IPTG and in the presence of 2% xylose (w/v); amounts of total RNA were 200 ng in lanes 1–24, 26 and 29, 100 ng in lanes 25 and 28, and 400 ng in lanes 27 and 30. P_xyl rnpA: presence (+) or absence (−) of a xylose-inducible plasmid-encoded B. subtilis rnpA gene. Lanes 1–12 and 25–27: primers specific for S. aureus rnpB (rnpB); lanes 13–24 and 28–30: primers specific for the mRNA encoding B. subtilis ribosomal protein S18 (S18). AMV: presence (+) or absence (−) of reverse transcriptase. For details on RT-PCR, see the Material and Methods section. Lanes 25–30 document that the amount of RT-PCR product was sensitive to RNA template concentration. The figure illustrates a representative experiment, but the results shown here were reproduced in five individual experiments using three independent total RNA preparations.

Figure 4.

Analysis of folding equilibria for wt and C258/C259 mutant B. subtilis P RNAs by native PAGE. RNAs (50 fmol, 5′-endlabeled) were preincubated either in buffer F containing 2 mM (F2) or 10 mM (F10) Mg²⁺, or in buffer KN containing 2 mM (KN2) or 4.5 mM (KN4.5) Mg²⁺ in a volume of 4–5 µl (for buffer F and KN compositions, see Materials and Methods). Lanes 1–3: no preincubation (kept at 4°C); lanes 4–6: preincubation of P RNAs for 70 min at 37°C; lanes 7–9: preincubation of P RNAs for 55 min at 37°C, addition of 1 µl B. subtilis P protein (final concentration 37 nM) and further incubation for 15 min at 37°C; lanes 10–12: as in lanes 7–9, but preincubation of P RNAs for 5 min at 55°C and 50 min at 37°C. Samples were run on 11.25% polyacrylamide gels in 1× THE buffer supplemented with 100 mM NH₄OAc and either 2 mM (F2, KN2) or 10 mM (F10, KN4.5) MgCl₂.

Figure 5.

5′-RACE analysis of B. subtilis trnSL-Ala1 3′-precursors. (A) Secondary structure of tRNA^Ala1 primary transcripts with RNase P and Z cleavage sites indicated; the arrow aligning the transcription terminator and tRNA acceptor stem indicates the position of the primer (‘180’) used for the 5′-RACE experiment; the boxed nucleotides (U74, A75) are replaced with the two C residues after RNase Z cleavage and CCA addition. (B) Analysis of 5′-RACE RT-PCR products on a native 8% PAA gel; M, 10-bp ladder as size marker; in lanes 1–3, primer ‘180’ (see above) and in lanes 4–6  a primer (see the Materials and Methods section) specific for T. thermophilus tRNA^Gly (Figure 1D) was used. The arrow on the left indicates the main amplification product obtained with primer ‘180’; its size was expected for a tRNA^Ala1 processing intermediate with a mature 5′-end but carrying the entire 3′-extension as in the primary transcript; this type of product was only detected in total RNA isolated from SSB320 cells grown in the absence of IPTG (lanes 1 and 2), but not in the presence of IPTG (lane 3); before reverse transcription, total RNA preparations were treated (+TAP) or not treated (− TAP) with tobacco acid pyrophosphatase (TAP). In lanes 5 and 6, total RNA from SSB320 cells grown in the absence of IPTG was supplemented with in vitro transcribed ptRNA^Gly (Figure 1D; 0.05 A₂₆₀ units ptRNA^Gly added to 1.0 A₂₆₀ unit of total cellular RNA) starting with 5′-pppG. A product corresponding to the size of ptRNA^Gly was only detected after TAP treatment (lane 6), but not without TAP treatment (lane 5) or without addition of in vitro transcribed tRNA^Gly (lane 4), thus confirming that TAP was active.