The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction

The debranching reaction is inhibited by branchpoint A-to-G mutation. (A) Splicing reactions were carried out with wild-type, ACAC, brG, or G1A pre-mRNA. (WT) wild type. (B,C) Spliceosomes assembled on brG (B) or G1A (C) pre-mRNA in Prp16-depleted Cwc25-HA extracts were individually precipitated with anti-HA antibody and incubated at 25°C for 1 h in 10 mM Tris-HCl, pH 8.8, or with 0 mM, 0.01 mM, 0.1 mM, 1 mM, 4 mM, 8 mM, or 20 mM MgCl₂ in the absence of (lanes 3–9) or presence of 150 mM KCl (lanes 10–16). (R) 1/20 (B) or 1/100 (C) of reaction mixture. (SP) spliceosome.