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

Inhibition of the debranching reaction by N-terminally tagged Cwc25 and Yju2. (A) Splicing was carried out in Prp16-depleted 4V5-Cwc25 or Cwc25-4V5 extracts. The spliceosome was precipitated with anti-V5 antibody, and then incubated at 25°C for 1 h in 10 mM Tris-HCl, pH 8.8, with 1 mM, 4 mM, or 8 mM MgCl₂ in the absence (lanes 3–5, 11–13) or presence of 150 mM KCl (lanes 6–8, 14–16). (B) Splicing was carried out in Prp16-depleted (lanes 1–4) or in both Yju2- and Prp16-depleted Cwc25-HA extracts supplemented with recombinant His-Yju2 (lanes 5–8) or Yju2-His protein (lanes 9–12). The spliceosomes were immunoprecipitated with anti-HA antibody, and then incubated in 10 mM Tris-HCl, pH 8.8, 8 mM MgCl₂ (lanes 3,7,11), or in 10 mM Tris-HCl, pH 8.8, 1 mM MgCl₂, 150 mM KCl (lanes 4,8,12). (R) 1/10 of reaction mixture, (SP) spliceosome, (D) DBR.