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