FIGURE 3.

Complete PICs challenged with Gdown1 show no defect at initiation and give rise to elongation complexes that are fully responsive to TFIIF after high salt rinse. PICs were prepared on the CMV template as described under “Experimental Procedures” and 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 before initiation. After a 1-min pulse with limiting, labeled NTPs, EECs were high salt-rinsed and chased in the presence or absence of 166 fmol of TFIIF for 30 or 60 s as described under “Experimental Procedures.” The space between lanes 5 and 6 separates non-adjacent lanes within the same gel. Lengths are shown to the left for the size markers in the left-most two lanes. A schematic of the assay is shown to the right of the gel.