FIG. 1.

Kinetic trap assay to test commitment to alternative 5′ splice site pairing. (A) Schematic of kinetic trap assay to test commitment to alternative 5′ splice site pairing. The common 3′ splice site of fruF competes for the female-specific 5′ splice site (proximal) or the cryptic 5′ splice site (distal). Reaction preferences and expected outcomes in the presence or absence of Tra/Tra2 are indicated. The black boxes within the exon denote Tra/Tra2 binding sites. (B) Native agarose gel electrophoresis of fruF incubated for 0 or 30 min in nuclear extract depleted of ATP. (C) Spliceosomal complex progression of fruF after addition of ATP and creatine phosphate to fruF preincubated in nuclear extract depleted of ATP. The last lane shows complex formation of fruF incubated in nuclear extract depleted of U4 snRNA for 30 min.