Full functional loading of Gdown1 to EECs takes nearly five minutes.
(A) PICs were assembled with TFIIF containing full-length RAP74 in solution on the AdML 31g(M) template  and EECs stalled at +30 were generated with ATP, UTP, and [α-32P]CTP as described in Materials and Methods. EECs were then incubated with 240 fmol of Gdown1 for 0 to 10 min (except lanes 1 and 2) and chased (except lane 1) for 30 sec with 200 μM NTPs. 240 fmol of Gdown1 is a 20-fold excess over Pol II and a 2-fold excess over TFIIF. DNA size markers were run in the far left lane of the gel and their lengths are indicated. Lengths of RNAs in the chase reactions were quantified as TFIIF-dependent and TFIIF-independent as noted on the right. A schematic of the assay is shown to the lower right of the gel. (B) Average percents of TFIIF-dependent transcription were quantified from results like those shown in panel (A) as described in Materials and Methods. The error bars indicate the mean ± S.D. based on five replicates.