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.