In vitro and in vivo investigations on the interaction of bacterial RNase P with tRNA 3’-CCA

The Ribonuclease P (RNase P) is a ribonucleoprotein enzyme, which catalyses the 5’-maturation of precursor tRNAs. Bacterial RNase P consists of one RNA subunit (P RNA; encoded by rnpB; ~400 nt), and a protein subunit (P protein, encoded by rnpA; ~120 aa). In vitro under elevated salt concentrations the RNA subunit is catalytically active. However, under physiological conditions the protein subunit is essential for activity. Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences It could be demonstrated that structural type A and type B bacterial RNase P RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Even a single copy of E. coli rnpBwt integrated into the amyE site of the B. subtilis chromosome was sufficient for cell viability. The findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not critical for RNase P function in vivo, at least under standard growth conditions. The precursor tRNA 3’-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3’-CCA termini (G292-C75 and G293-C74). Here, the phenotyes associated with disruption of the G292-C75 or G293-C74 pair in vivo was analyzed. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which can be infered to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about 2-fold, but 13-24-fold at 2 mM Mg2+. Moreover, the findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity or RNA degradation. However, native PAGE experiments revealed non-identical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, it cannot be excluded that altered folding of the mutant RNAs may have also contributed to their in vivo defect. In vivo role of bacterial type B RNase P interaction with tRNA 3’-CCA It has been unclear if catalysis by bacterial type B RNase P involves a specific interaction with p(recursor)tRNA 3’-CCA termini. We show that point mutations at two guanosines in loop L15 result in growth inhibition, which correlates with an enzyme defect at low Mg2+. For Bacillus subtilis RNase P, an isosteric C259-G74 bp fully and a C258-G75 bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base-pairing to tRNA 3’-CCA and emphasizing the importance of G258 identity. We infer the defect of the mutant enzymes to be primarily on the level of recruitment of catalytically relevant Mg2+, with a possible contribution from altered RNA folding. Cell viability of bacteria expressing mutant RNase P RNAs could be (partially) restored by RNase P protein overexpression, resulting in increased cellular RNase P levels. Finally, we demonstrate that B. subtilis RNase P is able to cleave CCA-less ptRNAs in vivo. We conclude that the in vivo phenotype upon disruption of the CCA interaction is either due to a global deceleration in ptRNA maturation kinetics or severe blockage of 5’-maturation for a subset of ptRNAs

The precursor tRNA 3′-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo

The L15 region of Escherichia coli RNase P RNA forms two Watson–Crick base pairs with precursor tRNA 3′-CCA termini (G292-C(75) and G293-C(74)). Here, we analyzed the phenotypes associated with disruption of the G292-C(75) or G293-C(74) pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G(74) base pair, but not a C292-G(75) pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg(2+) concentrations (2 mM), which we infer to be on the level of catalytically important Mg(2+) recruitment. At 4.5 mM Mg(2+), activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg(2+). Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect

In vitro and in vivo investigations on the interaction of bacterial RNase P with tRNA 3’-CCA

The Ribonuclease P (RNase P) is a ribonucleoprotein enzyme, which catalyses the 5’-maturation of precursor tRNAs. Bacterial RNase P consists of one RNA subunit (P RNA; encoded by rnpB; ~400 nt), and a protein subunit (P protein, encoded by rnpA; ~120 aa). In vitro under elevated salt concentrations the RNA subunit is catalytically active. However, under physiological conditions the protein subunit is essential for activity. Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences It could be demonstrated that structural type A and type B bacterial RNase P RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Even a single copy of E. coli rnpBwt integrated into the amyE site of the B. subtilis chromosome was sufficient for cell viability. The findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not critical for RNase P function in vivo, at least under standard growth conditions. The precursor tRNA 3’-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3’-CCA termini (G292-C75 and G293-C74). Here, the phenotyes associated with disruption of the G292-C75 or G293-C74 pair in vivo was analyzed. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which can be infered to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about 2-fold, but 13-24-fold at 2 mM Mg2+. Moreover, the findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity or RNA degradation. However, native PAGE experiments revealed non-identical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, it cannot be excluded that altered folding of the mutant RNAs may have also contributed to their in vivo defect. In vivo role of bacterial type B RNase P interaction with tRNA 3’-CCA It has been unclear if catalysis by bacterial type B RNase P involves a specific interaction with p(recursor)tRNA 3’-CCA termini. We show that point mutations at two guanosines in loop L15 result in growth inhibition, which correlates with an enzyme defect at low Mg2+. For Bacillus subtilis RNase P, an isosteric C259-G74 bp fully and a C258-G75 bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base-pairing to tRNA 3’-CCA and emphasizing the importance of G258 identity. We infer the defect of the mutant enzymes to be primarily on the level of recruitment of catalytically relevant Mg2+, with a possible contribution from altered RNA folding. Cell viability of bacteria expressing mutant RNase P RNAs could be (partially) restored by RNase P protein overexpression, resulting in increased cellular RNase P levels. Finally, we demonstrate that B. subtilis RNase P is able to cleave CCA-less ptRNAs in vivo. We conclude that the in vivo phenotype upon disruption of the CCA interaction is either due to a global deceleration in ptRNA maturation kinetics or severe blockage of 5’-maturation for a subset of ptRNAs

Analysis of folding equilibria for wt and C258/C259 mutant P RNAs by native PAGE

<p><b>Copyright information:</b></p><p>Taken from " and investigation of bacterial type B RNase P interaction with tRNA 3′-CCA"</p><p></p><p>Nucleic Acids Research 2007;35(6):2060-2073.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874595.</p><p>© 2007 The Author(s)</p> RNAs (50 fmol, 5′-endlabeled) were preincubated either in buffer F containing 2 mM (F2) or 10 mM (F10) Mg, or in buffer KN containing 2 mM (KN2) or 4.5 mM (KN4.5) Mg 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 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 NHOAc and either 2 mM (F2, KN2) or 10 mM (F10, KN4.5) MgCl

Growth curves of SSB318 cells complemented with pHY300 derivatives, carrying wt (squares) or C238 (triangles with apex at the top) or C239 (triangles with apex at the bottom) mutant alleles in the presence (+) or absence (−) of IPTG

<p><b>Copyright information:</b></p><p>Taken from " and investigation of bacterial type B RNase P interaction with tRNA 3′-CCA"</p><p></p><p>Nucleic Acids Research 2007;35(6):2060-2073.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874595.</p><p>© 2007 The Author(s)</p> The better growth of SSB318 bacteria expressing wt (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 gene in the SSB318 mutant strain is weaker than expression from the native promoter in the original strain W168 used to construct SSB318 ((), therein). Improved growth was also observed when we expressed wt from pHY300 in strain SSB318 (data not shown), showing that this effect is not specific for . We conclude that plasmid-borne expression of or wt saturates the cellular RNase P levels in SSB318 bacteria and thereby restores wild-type-like growth

Secondary structure illustrations of () (type B), () (type B) and () (type A) RNase P RNA according to ()

<p><b>Copyright information:</b></p><p>Taken from " and investigation of bacterial type B RNase P interaction with tRNA 3′-CCA"</p><p></p><p>Nucleic Acids Research 2007;35(6):2060-2073.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874595.</p><p>© 2007 The Author(s)</p> The two G residues in L15, known () or suspected () to be involved in the interaction with tRNA 3′-CCA, are highlighted by gray ovals. () Proposed interaction of a canonical bacterial ptRNA ( ptRNA) with the L15 loop of 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)

Radioactive reverse transcription PCR (RT-PCR) analysis of strain SSB318 complemented with wt or C238/C239

<p><b>Copyright information:</b></p><p>Taken from " and investigation of bacterial type B RNase P interaction with tRNA 3′-CCA"</p><p></p><p>Nucleic Acids Research 2007;35(6):2060-2073.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874595.</p><p>© 2007 The Author(s)</p> PCR products were analyzed on a 10% polyacrylamide/8 M urea gel. Lanes 1–30: total RNA from SSB318 complemented with wt (lanes 1–4 and 13-16), C238 (lanes 5–8, 17–20 and 25–30) or C239 (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 : presence (+) or absence (−) of a xylose-inducible plasmid-encoded gene. Lanes 1–12 and 25–27: primers specific for ; lanes 13–24 and 28–30: primers specific for the mRNA encoding 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