FIG. 3.

p65 cross-linking occurs before A complex formation and declines later. RNAs were incubated with 60% HeLa nuclear extract, and MB cross-linking was carried out at 15 and 180 min. X23H is the natural intron between TM exons 2 and 3; spliceosome assembly is blocked at the A complex stage because of the proximity of the branch point (shown schematically as the solid circle) to the 5′ splice site. HP1 is derived from GC+DX by insertion of a stable hairpin between the polypyrimidine tract (shown as the solid rectangle) and 3′ splice site, which blocks step 2 of splicing. Lane M, protein size markers (kilodaltons). p65 cross-linking with HP1 was identical to that with GC+DX (compare lanes 1 and 2 with 5 and 6), whereas with X23H, p65 cross-linking occurred but did not subsequently decline (lanes 3 and 4).