FIG 5.

Synergy requires the cleavage-promoting activity of TFIIS. (A) A runoff assay was performed as described in the legend to Fig. 2A. Fifty femtomoles of wild-type TFIIS (lanes 2 to 5 and 6 to 9), TFIIS with point mutations (D290A and E291A; TFIIS Ac), or TFIIS with deletion of residues 290 and 291 (290Δ and 291Δ; TFIIS del) and 0.5 μg of Ccr4-Not complex were added as indicated. Runoff was initiated by adding GTP and UTP. Reactions were stopped at 0 s (lanes 2, 6, 10, and 14), 30 s (lanes 3, 7, 11, and 15), 60 s (lanes 4, 8, 12, and 16), and 120 s (lanes 5, 9, 13, and 17) after the addition of the nucleotides, and transcripts were analyzed on a 10% urea denaturing gel. (B) The percentage of runoff products was calculated and plotted over time.