Figure 4.

Effects of rhTRF2 and flag⋅hTRF2–TFIIA on transcription by RNA polymerase II. (A) Basal transcription from the AdML promoter. System A contained 15 ng of rhTFIIB, 20 ng of rhTFIIE, 20 ng of rhTFIIF, 0.3 μl of purified TFIIH, and 0.7 μl of purified RNA polymerase II. Transcription was performed in system A alone (lane 1) with 5 ng of rhTBP (lane 2), 10 ng of rhTRF2 (lane 3), or 40 ng of flag⋅hTRF2–TFIIA complex (containing 10 ng of hTRF2; lane 4). (B) Basal transcription from the Hsp70 promoter. Reactions contained in addition to system A: 5 ng of rhTBP in lanes 2 and 5–10; 1 μl of flag⋅TFIID (containing 5 ng of TBP) in lanes 11–16; 10 ng of rhTRF2 in lanes 3, 6, 9, 12, and 15; 40 ng of purified flag⋅hTRF2–TFIIA complex (10 ng of flag⋅hTRF2) in lanes 4, 7, 10, 13, and 16; 0.5 μl of Ni-NTA Agarose-purified TFIIA in lanes 8–10 and 14–16. (C) Activated transcription from the E4 promoter. System B contained 30 ng of Gal4-VP16, 150 ng of rhPC4, 12 ng of rhTFIIA, 10 ng of rhTFIIB, 20 ng of rhTFIIE, 25 ng of rhTFIIF, 1 μl of flag⋅TFIID, 0.5 μl of TFIIH, and 0.5 μl of RNA polymerase II. Reactions were supplemented with the following: 12 and 36 ng of rhTFIIA in lanes 2 and 3; 12 and 36 ng of Ni-NTA Agarose-purified TFIIA in lanes 4 and 5 and 10 and 11; 6 and 18 ng of rhTRF2 in lanes 6 and 7 and 10 and 11; 24 and 72 ng of purified flag⋅hTRF2–TFIIA complex (6 and 18 ng of flag⋅hTRF2) in lanes 8 and 9. (D) Transcription from the E4 promoter in the presence of NC2. Lanes 1–11 contained system B, which is described in C. Lanes 2–11 were supplemented with 20 ng of purified NC2. Other additions were as follows: 12 and 36 ng of rhTFIIA in lanes 3 and 4; 12 ng of Ni-NTA Agarose-purified TFIIA in lane 5; 36 ng of Ni-NTA Agarose-purified TFIIA in lanes 6 and 11; 6 and 18 ng of rhTRF2 in lane 7 and lanes 8 and 11, respectively; 24 and 72 ng of purified flag⋅hTRF2–TFIIA complex (containing 6 and 18 ng of flag⋅hTRF2, respectively) in lanes 9 and 10.