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. 2014 Mar 4;289(16):11143–11152. doi: 10.1074/jbc.M113.544395

FIGURE 7.

FIGURE 7.

Pol IIG is inefficient in supporting transcription on a bubble template, but Gdown1 does not affect transcription when added to an existing bubble template PIC. A, PICs were formed on premelted templates using only a subset of GTFs (TBP, TFIIB, TFIIF) as described under “Experimental Procedures.” Gdown1 was either preincubated in 20-fold excess over pol II (pre) or added in 20-fold excess over pol II to the already assembled PIC (post), followed by pulse labeling as described under “Experimental Procedures”. The amount of Gdown1 (240 fmol) corresponds to 3.3-fold excess over TFIIF (72 fmol). Lane 1 contained all components for PIC formation except TFIIF to demonstrate TFIIF dependence for the initiation assay. The length of the size marker is shown on the left. B, as in A, except all GTFs were used. C, as described in B with Gdown1 pretreatment of 20-fold excess over pol II (pre) or with 0.62 pmol of IIF (8× standard amount of TFIIF used) incubated with Gdown1 and pol II during the pretreatment. treat., treatment.