Fig. 4

TFIIEα and CAK releases and Pol II phosphorylation occur before DNA opening. a Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF and rIIH/WT, in presence of either ATP (200 μM) or ATPγS (200 μM). After washes, the binding of various factors has been evaluated by immunoblotting. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. b Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, the Core-TFIIH, XPD/WT, and the CAK, in presence of ATP (200 μM). After washes, immunoblot analysis was done for different proteins. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. c Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and rIIH/WT, in presence of ATP (200 μM) and THZ1 (1 and 10 μM). The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. d When indicated (+), biotinylated AdMLP bound to streptavidin magnetic beads were preincubated 20min (at 25 °C) with the general transcription machinery (Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and rIIH/WT) in presence of Triptolide (TPL, 10 μM) before addition of ATP (200 μM). The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. e Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, and TFIIF, in presence of ATP (200 μM) and either rIIH-XPB/WT or /Fs740. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. f Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, and TFIIF, in presence of ATP (200 μM) and either rIIH-XPB/WT or /K346R. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. Source data are provided as a Source Data file