Spliceosomal factor requirements for step 2 catalysis of Bact spliceosomes assembled on actin pre-mRNAs. (A) Extent of splicing in vitro catalyzed by affinity-purified spliceosomes assembled on the Actwt pre-mRNA substrate. The Bact complex (lane 1) was affinity-purified by a three-step procedure (see Supplemental Methods). Peak fractions of the second glycerol gradient were used for complementation assays with recombinant proteins at a 30-fold molar excess over the Bact complex (10 fmol) as indicated (lanes 2–9). Reaction mixtures were incubated (45 min at 23°C) in the presence of ATP. RNAs were extracted, separated by denaturing polyacrylamide gel electrophoresis (PAGE), and visualized by autoradiography. (B) Bact complexes assembled on Act7 pre-mRNA were purified and eluted from the amylose matrix. Subsequently, the complexes were complemented with the recombinant proteins indicated and were further incubated in the presence of ATP. Symbols for pre-mRNA, splicing intermediates, and products are indicated on the right: the 5′ exon (with the three MS2 loops shown) in gray, intron as a thin black line, 3′ exon in black. (*) Uncharacterized pre-mRNA bands.
