FIGURE 5.

In a continuous transcription assay, Gdown1 does not inhibit stimulation of elongation by TFIIF until complexes are actively transcribing. PICs were prepared on the CMV template as described under “Experimental Procedures.” A, PICs were incubated with 240 fmol of Gdown1 (20-fold excess relative to pol II and 3.3-fold excess relative to TFIIF used to assemble PICs) or buffer. In lanes 7–12, the supernatant, including the unbound Gdown1, was removed before the pulse (inclusion of bracketed step in the schematic). PICs were then pulse-labeled and chased with no rinse step. In the lanes where the supernatant had been removed (lanes 7–12), additional (add'l) TFIIF (166 fmol) was added with the chase. The schematic of the assay is shown to the right, and the lengths of the size markers are shown to the left. B, as in the left of A, except in lanes 7–12, the large subunit of TFIIF was truncated to only the 227 N-terminal amino acids. Lengths of size markers are shown to the right.