FIG 2.

Ccr4-Not and TFIIS synergize to rescue arrested RNAPII. (A) Runoff transcription assay in the presence of TFIIS and Ccr4-Not. Arrested RNAPII elongation complexes (70-nt ECs) were formed in the presence of O-me-GTP, as described in the Materials and Methods section, for 10 min (−10, lane 1). Afterwards, 50 fmol of TFIIS or 0.5 μg of Ccr4-Not or both were added. After 10 min, GTP and UTP were added to produce runoff (RO) transcripts. 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. The transcripts were purified and analyzed on a 10% urea-polyacrylamide gel. (B) The percentage of runoff products from the gel in panel A was calculated and plotted over time. (C) Stalled elongation complexes were formed as described in the legend to panel A, except that the O-me-GTP transcription terminator was not added to the reaction mixture. Approximately 50 fmol of purified TFIIS (lanes 2 to 5 and 10 to 13) or 0.5 μg of the Ccr4-Not complex (lanes 6 to 9 and 10 to 13) was added to the transcription reaction mixtures and incubated for 10 min. The transcription reaction was then resumed with the addition of GTP and UTP. Reactions were stopped at 0, 30, 60, and 120 s, and transcripts were purified and analyzed on a 10% denaturing gel. (D) The percentage of runoff products was calculated and plotted as a function of time.