Fig. 3

Defective TFIIE and TFIIH alter the preinitiation complex. a Biotinylated AdMLP bound to streptavidin magnetic beads was incubated with Pol II, TFIIA, TFIIB, TFIID (TBP), TFIIF, and TFIIH, in the presence (+) of either rIIEαβ/WT, rIIEαβ/A150P, or rIIEαβ/D187Y. After washes, the binding of different factors was evaluated by immunoblotting. The loading proteins used for PIC formation and the different rIIEs (in the following order rIIEαβ/WT, rIIEαβ/A150P, and rIIEαβ/D187Y) are indicated at the right part of the panel (load). The signals for IIEα, IIEβ, XPB, XPD, and CDK7 were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. b Immunoprecipitated rIIH/WT (using anti-p62) was incubated with rIIEs at 150 and 500 mM KCl. After washes, the coimmunoprecipitated proteins were resolved by SDS–PAGE and blotted with anti-XPB, anti-IIEα, and anti-IIEβ. Graph shows in arbitrary units (a.u.) the ratio IIEα/IIEβ for each rIIE. The results are representative of three independent experiments. c Pol II, TFIIA, TFIIB, TFIID (TBP), and TFIIF were incubated with biotinylated AdMLP bound to streptavidin beads in presence (+) of rIIEα/WT, rIIEβ/WT, and TFIIH. Immunoblot analysis has been done with different antibodies, as indicated. d TFIIH was immunoprecipitated with anti-p62 and incubated (+) with rIIEα/WT and/or rIIEβ/WT. After washes, immunoblot analysis of TFIIH (XPD, p52, CDK7) and TFIIE (IIEα and IIEβ) has been done. e Recombinant rIIEα/WT, co-produced with either rIIEβ/WT (lanes 1 and 2), rIIEβ/A150P (lanes 3 and 4) or rIIEβ/D187Y (lanes 5 and 6), was immunoprecipitated at 150 and 500 mM KCl. After washes, the proteins were resolved by SDS–PAGE and blotted with antibodies against IIEα and IIEβ. Graph shows the ratio IIEβ/IIEα (n = 3, means ± s.d.) in arbitrary units (a.u.). The results are representative of three independent experiments. f rIIH containing either XPD/WT (lanes 1 and 2), XPD/R112H (lanes 3 and 4), or XPD/R722W (lanes 5 and 6) were immunoprecipitated with anti-XPD and incubated at 150 and 500 mM KCl. After washes, the bound proteins were resolved by SDS–PAGE. The immunoprecipitated signals (IP) for XPB, XPD, and CDK7 were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. g Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, the Core-TFIIH, the CAK and either XPD/WT, /R112H, or /R722W. After washes, immunoblot analysis of different factors has been done. The results (n = 3, means ± s.d.) are representative of three independent experiments. Source data are provided as a Source Data file