Experimental design and example of data. The Prp2-deficient spliceosomal complex BactΔPrp2 (stalled through its lack of Prp2) is catalytically activated by addition of Prp2 together with Spp2 and ATP. This leads to complex B*, which, on binding the step 1 factor Cwc25, catalyzes step 1 of splicing, generating complex C; for details, see Introduction. U2/5/6 snRNPs are shown as circles. The composition of the activated spliceosome is shown underneath; green stars indicate the spliceosomal proteins that were tagged with EGFP. (Inset) Release or recruitment of an EGFP-labeled protein (green star) from/to the spliceosome (carrying Atto647N, red star). The cross-correlation amplitude at short correlation time τ is proportional to the amount of doubly labeled spliceosomes.
