Figure 7.

Manganese specificity and pH dependence of debranching. (A) Increasing amounts of Dbr1 as specified were incubated with 10 nM of 5′-labeled substrate and either 4 mM MnCl₂ or 2.5 mM MgCl₂ for 10 min at 22°C. The percentage of the substrate that was debranched is plotted as a function of input Dbr1. (B) Manganese titration. Reaction mixtures containing 50 mM Tris–HCl (pH 7.0), 2.5 mM DTT, 25 mM NaCl, 0.01% Triton X-100, 0.1 mM EDTA, 0.15% glycerol, 1 nM Dbr1 and 10 nM branched substrate and manganese as specified were incubated for 10 min at 22°C. (C) pH dependence. Reaction mixtures containing 50 mM Tris buffer [either Tris–acetate for (pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5) or Tris–HCl (pH 7.0, 7.5, 8.0, 8.5)], 4 mM MnCl₂, 2.5 mM DTT, 25 mM NaCl, 0.01% Triton X-100, 0.1 mM EDTA, 0.15% glycerol 10 nM RNA and 1 nM Dbr1 were incubated for 10 min at 22°C. The extent of debranching is plotted as a function of pH. The branched RNA substrate used for these experiments is depicted in Figure 6B.