FIGURE 2.
ATP-dependent Prp16 activity is sufficient to generate stable, catalytically activated C* complexes in the absence of Slu7, Prp18, and Prp22. (A) Bact complexes were purified and complemented with Prp2, Spp2, and Cwc25 on the amylose matrix to generate C complexes. One-half of the reaction mixture was supplemented with buffer, the other half with Prp16 and ATP to generate C* complexes. After incubation, complexes were washed with GK75 buffer and eluted with maltose. Complexes (specific activity 100 cpm/fmol) were sedimented on glycerol gradients containing 75 mM KCl. Fractions were analyzed by Cherenkov counting. (B) Fraction 15 of both complexes was analyzed directly (lanes 2,6) or was additionally complemented with Slu7/Prp18 and Prp16 +/− ATP as indicated. After incubation, RNAs were extracted, separated by denaturing PAGE, and visualized by autoradiography. RNA species (right), see Figure 1.