FIGURE 3.
Displacement of the step 1 factor Cwc25 from the spliceosome by Slu7/Prp18 as analyzed by dcFCCS. (A) Affinity-purified Bact complexes assembled on Atto-Actwt pre-mRNA were complemented on the matrix with Prp2, Spp2, ATP, and Cwc25-Alexa. The reaction mixture was incubated and then depleted of ATP. After washing of the matrix, reconstituted C complexes were eluted with maltose and complemented in solution as indicated above each column, and dcFCCS measurements were performed. (B) Affinity-purified Bact complexes assembled on Atto-ActACAC 3′SS mutant (see text) were complemented on the matrix as described in A. The reaction mixture was incubated, C complexes were eluted and complemented in solution as indicated above each column, and dcFCCS measurements were performed. Affinity-purified Bact complexes assembled on Atto-Act7wt (C) and Atto-Act7ACAC 3′SS mutant (D) were complemented on the matrix to generate C complexes. Eluted C complexes were then complemented in solution as indicated above each column, and dcFCCS measurements were performed. Cross-correlation amplitudes derived from two independent experiments are shown for each complex. Error bars indicate the standard deviation from two independent measurements.