FIGURE 3.

Characterization of the debranching reaction. A splicing reaction was carried out in Prp16-depleted Cwc25-HA extracts (lane 1), and the spliceosome was precipitated with anti-HA antibody (lane 2). The spliceosome was incubated at 25°C for 1 h under various conditions: (A) 10 mM Tris-HCl, pH 8.8 and 4 mM MgCl₂ alone (lane 3) or with 50 mM, 100 mM, 150 mM, 200 mM, or 300 mM of KCl (lanes 4–8) or NaCl (lanes 9–13); (B) 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 (lanes 3–9) or presence of 150 mM KCl (lanes 10–16); (C) 10 mM Tris-HCl, pH 8.8, with 0 mM, 0.001 mM, 0.01 mM, 0.1 mM, 1 mM, 2 mM, 4 mM, or 8 mM MnCl₂ in the absence (lanes 3–8) or presence of 150 mM KCl (lanes 9–14). (R) 1/10 of reaction mixture, (SP) spliceosome.