1. Reconstitution of the Transcription Factor TFIIH
Franck Tirode, Didier Busso, Frédéric Coin, Jean-Marc Egly 
Molecular Cell 
Volume 3, Issue 1, Pages (January 1999)
DOI: /S (00)80177-X

2. Figure 1 The Recombinant Transcription Factors
(A) Overexpression of TFIIH subunits in baculovirus-infected Sf9 cells. Five microliters of each infected cell extract as indicated at the top of the figure (lanes 2–7) and 20 μl of HeLa TFIIH (lane 1) were loaded on a 12% SDS-PAGE and immunoblotted (WB) with TFIIH antibodies.
(B) Analysis of recombinant CAK. 2.5 μg of either mutant (CAKm, lane 2) or wild-type (CAK, lane 3) kinase complex containing, in addition to a tagged cyclin H (t-CycH) and MAT1, either mutant or wild-type cdk7, respectively, was loaded on a 15% polyacrylamide gel and Coomassie stained (SDS-PAGE). Molecular weight markers: M, lane 1. Two hundred nanograms of both mutant and wild-type CAK and 5 μl of TFIIH were analyzed by Western blotting using monoclonal antibodies raised against the cdk7, cyclin H, and MAT1 subunits (WB) and tested for kinase activity in the presence of 1 μg of ctd as substrate and 0.3 pmol of [γ32P]ATP (kinase).
(C) Purified recombinant trancription factors. Highly purified (around 100 ng) TFIIB, TFIIEα, TFIIEβ, TFIIF, and truncated human TBP overexpressed in E. coli were loaded on a 12% SDS-PAGE and Coomassie stained.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

3. Figure 2 Purification of Recombinant rIIH6
Sf9 cells were coinfected with baculoviruses encoding for each of the subunits that form the core TFIIH/XPD, and the extracts were successively loaded on a heparin ultrogel and a metal chelate affinity column. After extensive washing with a buffer containing 0.4 M KCl (lanes 2–4), the proteins were eluted from a heparin ultrogel column with the same buffer containing 0.5 M KCl (lanes 5–7). The first two fractions (0.5/1 and 0.5/2; lanes 5 and 6) were then loaded on a metal affinity column, and proteins were eluted with buffer containing 10 mM imidazol (lanes 9–11) and 100 mM EDTA (lane 12). Ten microliters of each fraction was tested in a runoff transcription assay (Transcription) and analyzed for the presence of TFIIH subunits by Western blotting (WB); (lane 1) IIH, HeLa-TFIIH (1 μl). The 309 nt runoff transcript and TFIIH subunits are indicated.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

4. Figure 3 Enzymatic Activities of the rIIH Complexes
Equivalent amounts (with XPB polypeptide as reference) of rIIH9, rIIH6, and rIIH5 recombinant factors overproduced and purified as previously stated, containing either nine, six (core TFIIH/XPD), or five (the core) subunits, respectively, were analyzed by silver-stained SDS-PAGE and tested for helicase, ATPase, and transcription activities. The helicase and the ATPase assays were performed upon immobilization on protein G–agarose beads cross-linked with antibody raised against p44 (Coin et al., submitted). When indicated, HeLa TFIIH (IIH) is used as a qualitative control. Minus, assays performed in the absence of TFIIH; triangle, heated helicase template. The 5′ to 3′ or the 3′ to 5′ polarity of the helicase, the Pi generated upon ATP hydrolysis, the nature of the polypeptides, as well as the size of runoff transcript are indicated at the right of each panel. Quantification of the transcription activity (lowest panel) of the three recombinants, rIIH9, rIIH6, and rIIH5, was done using a Bio-imaging Analyzer FUJIX BAS 2000.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

5. Figure 4
The Five Subunits of the Core TFIIH Are Required for Transcription
Equal amounts of partially purified IIH complexes from Sf9 coinfected cells, containing either the nine subunit (rIIH9) or the six subunits of core TFIIH (rIIH6) or lacking one of the six subunits (lower panel) as indicated at the top of each panel, were tested for transcription activity in an S1 nuclease assay (upper panel). The S1 oligonucleotide probe (60 nt) and the 41 nt digested oligonucleotide are indicated at the right of the panel. Controls (lane 10 and 11, upper panel) represent the radiolabeled probe either diluted and undigested or digested with 100 U of S1 nuclease. IIH is the HeLa TFIIH control.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

6. Figure 5
Requirement of XPB and XPD Helicase Activities for In Vitro Transcription
Equivalent amounts, according to Western blot estimation, of recombinant wild-type rIIH6 or mutated rIIH6/XPB-K346R and rIIH6/XPD-K48R, as well as rIIH6/p44c and rIIH6/p44t containing either form of p44 differing by three amino acid changes, were tested in an in vitro runoff transcription assay. I–VI indicate the helicase motifs of XPB and XPD, whereas hatched boxes indicate the cysteine-rich motifs. The amino acid changes are shown. Minus, transcription without TFIIH.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

7. Figure 6 Role of CAK in the Transcription Reaction
(A) rIIH6 (5 μl) in addition to wild-type rCAK (100 ng) or rCAKm (mutated in the kinase motif of cdk7) was tested in a transcription assay using either Ad2ML or DHFR promoter to produce 309 nt and 200 nt runoff transcripts, respectively. Quantification of the transcription activity was done using a Bio-imaging Analyzer FUJIX BAS As a qualitative control, HeLa TFIIH was used (IIH). Minus, transcription without TFIIH.
(B) rIIH5 (10 μl) and either rCAK (50 and 100 ng) or highly purified HeLa CAK (2.5 and 5 μl, corresponding to the same specific activity toward ctd; Rossignol et al. 1997) or HeLa-purifed CAK/XPD were tested as indicated in (A) using Ad2MLP.
(C) The phosphorylation of RNA pol II was tested in an in vitro transcription assay containing all the basal transcription factors and the recombinant subcomplexes rIIH6, rCAK, rCAKm, and CAK/XPD as indicated at the top of the figure. Quantification of the RNA pol IIO (the hyperphosphorylated form) was performed using Molecular Analyst software (Bio-Rad).
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)

8. Figure 7 CTD Phosphorylation Does Not Require Active TFIIH
(A) In vitro transcription in a reconstituted system was performed from AdML promoter either in its wild-type form (WT) or in an open form (−8/+2), in the presence of either TFIIH or a reconstituted complex (rIIH4) lacking both XPB and XPD helicases.
(B) Equivalent amounts (5 or 10 μl, as indicated at the top of the panel) of either wt rIIH6, rIIH6/XPB-K346R, or rIIH6/XPD-K48R mutated recombinant complexes (see also at the top of Figure 5) were tested in an in vitro transcription assay for their capacity to be stimulated by rCAK (50 ng).
(C) The phosphorylation of RNA pol II was tested in an in vitro transcription assay (as in [B]) containing all the basal transcription factors and either rIIH6, rIIH6/XPD-K48R, or rIIH6/XPB-K346R in the presence or absence of rCAK as indicated at the top of the figure. Quantification of the RNA pol IIO was done as in Figure 6C. (D) Promoter-opening experiments using KMnO4 footprinting were done with 5 μl of either recombinant wt rIIH6 (lane 2) or ATPase site-mutated rIIH6 complexes (lanes 3 and 4), in addition to 50 ng of rCAK. Pattern comparisons of the KMnO4 experiments using Molecular Analyst software (Bio-Rad) are shown on the right of the figure. Minus, experiment without TFIIH. The promoter sequence and the opened region are indicated.
(E) Phosphorylation of the RNA pol II was perfomed as in (C), in the presence of rCAK, except that dATP was the only nucleotide added.
Molecular Cell 1999 3, 87-95DOI: ( /S (00)80177-X)