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

Abstract

<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