Figure 6.

Transcription activity of the rIIHs. (A) Basal transcription activity of the rIIHs. The purified rIIHs were added to an in vitro reconstituted transcription system lacking TFIIH. The length of the corresponding transcript is indicated on the left side. The transcription activity of all variants was assessed using increasing amounts of rIIHs for 30 min. The signals were quantified and plotted in arbitrary units (au). The results are representative of three independent experiments. The complete gel is shown in Fig. S2 A. (B) Phosphorylation of the RNA polymerase II during in vitro reconstituted transcription assays. The RNA pol II kinase activity of low salt immunopurifed rIIHs was analyzed in an in vitro assay containing all the basal transcription factors and the AdMLP. Arrows indicate hypo (IIA) and hyper (IIO) phosphorylated forms of RNA pol II. The results are representative of two independent experiments. The complete gel is shown in Fig. S2 B. (C) In vitro phosphorylation of the GST-CTD fusion protein was performed with equal amounts of rIIHs in the presence of 0.14 µM [γ-³²P] ATP. Coomassie blue–stained gel (staining; top) and autoradiography (autoradio; bottom) of the incubated fractions are shown. The results are representative of three independent experiments. The complete gels are shown in Fig. S2 C. (D) In vitro reconstituted transcription assays were performed following the protocol scheme. rIIH XPD/R683W, rIIH XPD/Q452X, /199insPP, or /I455del were preincubated either alone or in combination, in the presence of RNA pol II, the general transcription factors (GTF), the AdMLP template, ATP, and CTP. 15 min later, the transcription process was initiated by addition of GTP and UTP. The reactions were performed for 15 and 30 min. The signals of three independent experiments were then quantified and plotted in arbitrary units (au).