Stimulation of the XPB ATP-dependent helicase by the beta subunit of T
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《核酸研究医学期刊》
Department of Chemistry and Biochemistry, The Molecular Biology Institute, University of California Los Angeles Los Angeles, CA 90095-1569, USA
*To whom correspondence should be addressed. Tel: +1 310 825 1620; Fax: +1 310 267 2302; Email: gralla@chem.ucla.edu
ABSTRACT
TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.
INTRODUCTION
Eukaryotic class II transcription requires at least 30 polypeptides for basal transcription and many more are needed for properly regulated transcription. These polypeptides include RNA polymerase II and components of the general transcription factors. TFIID and TFIIB recruit the polymerase and TFIIF to the promoter, and other factors modify the transcription complex to allow it to proceed into early elongation phase (1–3). In the step-wise model of assembly, TFIIE and TFIIH are added immediately after polymerase (4) and are used in the opening of the DNA and in the initial steps of RNA synthesis, including promoter escape (5–10). TFIIE and TFIIH are essential proteins in vivo (11,12). In vitro, TFIIE and TFIIH are required for these transcription steps, although their function can be partially bypassed on some supercoiled templates (13).
TFIIE is composed of two subunits, a larger 56 kDa subunit TFIIE and a smaller 34 kDa subunit TFIIE? (14,15). The TFIIE heterotetramer (2?2) has no known catalytic activity and recruits TFIIH to complete the assembly pathway (4,8,16,17). TFIIH is a large complex with 10 subunits and several enzymatic activities, including ATP hydrolysis, energy-dependent DNA unwinding and phosphorylation (18–24). Two subunits of TFIIH, XPB and XPD, can accomplish DNA unwinding, although the polarity of the process is different for each (20–22). The XPD ATP-dependent helicase is used to open DNA during repair, and its activity within TFIIH is strong owing to the activation by its partner subunit p44 (25,26). The XPB ATP-dependent helicase is used to open the promoter DNA during transcription and is also used in promoter escape (27–31). Its helicase activity within TFIIH is weaker and its p52 partner subunit does not enhance this activity (32). XPB and XPD have other roles that appear to be independent of helicase action. The ATPase activity of XPB is needed for the activation of the XPG endonuclease during nucleotide excision repair (33). XPD provides structural integrity to TFIIH (25,26), especially to the CAK kinase complex that is involved in the transition from transcription initiation to elongation (16,19,24,26,28,34–36) and other cellular processes (17,37).
Although TFIIE has no catalytic activities (14,15), it can influence those of TFIIH (16,24,36,38). It has been reported to positively regulate the ATPase and kinase activities of TFIIH (19) and negatively regulate its helicase activity (39). The negative regulation is specific to the XPB helicase (39). From these observations, it has been suggested that TFIIE inhibits the ability of TFIIH to open promoter DNA until the complete complement of general transcription factors is assembled, at which time this constraint may be relieved. Overall, TFIIE and TFIIH likely work together to modify the pre-initiation complex so as to allow the RNA polymerase to function in transcription initiation and to transit to early elongation. During this critical transition process, the mRNA becomes capped and TFIIH begins to catalyze the staged series of changes in the RNA polymerase CTD that sets the stage for coupled elongation and RNA processing events. Although TFIIE is required for this transition (19,36), its role has not been clearly established. The role of the beta subunit is of particular interest, as its C-terminus is crowded with interaction domains for other general transcription factors (36,38).
Both the alpha and beta subunits of TFIIE can interact with XPB (35) and it has been suggested that the alpha subunit is dominant for the inhibition of the XPB function (39). No specific role has emerged for the interaction of the beta subunit with XPB, although the possibility has been raised that it functions in the transition into early elongation phase (36,38). Because promoter opening and escape require the XPB ATPase and helicase activities (5,28,31,40), we have investigated the possible role of the TFIIE beta subunit in modulating these activities. Below, we show that the isolated beta subunit can stimulate the XPB helicase and ATPase activities and that such stimulation has a functional role in transcription.
MATERIALS AND METHODS
Protein expression and purification
The human XPB (ERCC3) clone was a gift from the J. -M Egly laboratory. Glutathione S-transferase (GST)-tagged XPB was expressed and partially purified as described previously (22,41) with the following modifications. An aliquot of 400 ml of Escherichia coli cells were induced with 0.1 mM of isopropyl-?-D-thiogalactopyranoside (IPTG) for 30 min at 37°C. The cell pellets were collected and re-suspended in 10 ml of 1x phosphate-buffered saline (PBS) with 1% TritonTM X-100. An aliquot of 150 μl of Glutathione Sepharose 4B was used in the batch purification protocol as described previously (Amersham Pharmacia Biotech). Each wash used 10 ml of 1x PBS with 1% TritonTM X-100 for a total of four washes. The XPB was eluted with 50 mM Tris–HCl (pH 7.9), 10 mM reduced glutathione and 50% glycerol. The eluted sample was dialyzed against 50 mM Tris–HCl (pH 7.9) and 50% glycerol to remove the reduced glutathione. GST was expressed and purified the same way.
Human TFIIE and TFIIE? clones were obtained from Dr K. Yokomori. Human TFIIE?, its C-terminal truncated mutants (stop codons were placed accordingly using QuikChangeTM site-directed mutagenesis kit from Stratagene) and point mutants with His6 tagged were expressed in 4–500 ml of E.coli BL21(DE3) cells in 0.3 mM IPTG, 2% glucose and Luria–Bertani for 1 h at 37°C after OD600 nm reached 0.5. Ni-based resin (Qiagen and Active Motive) was used for purification (see Qiagen's manual for purification procedures and solutions). Proteins were analyzed on 12% Tris/Glycine SDS–PAGE with the correct molecular weights and were >95% pure. The proteins contained low but detectable helicase activity.
Human TFIIE with a His6 tag was a gift from Hao Duong (UCLA, Department of Chemistry and Biochemistry). Human TFIIE and N-terminal truncated mutants with an N-terminal FLAG tag were cloned into pET11a (Novagen). They were expressed the same way as TFIIE? (see above) and were purified using ANTI-FLAG? M2-Agarose (Sigma, see the manufacturer's purification procedures and solutions). The proteins were eluted with FLAG peptide. They were analyzed on 10% Tris/Glycine SDS–PAGE and were >95% pure.
Human TFIIH was purchased from Protein One (http://www.proteinone.com).
Helicase and ATPase assays
Helicase assays were as described previously (21,22,39). Briefly, 20 ng of 32P-labeled oligonucleotide (5'-AAACGACGGCCAGTGAATTCGAGCTCGGTACCCGG-3') complementary to M13mp19+ was annealed to 1 μg of the single-stranded plasmid DNA in 25 mM NaCl and 2.5 mM MgCl2. The duplex substrate was then purified in a Sephadex 200 size exclusion column. An aliquot of 400–500 ng of duplex was recovered free of un-annealed labeled oligonucleotide. Helicase assays for XPB and TFIIE? or its mutants were incubated for 30 min at 37°C in the presence of 4 mM MgCl2, 4 mM ATP, 50 μg/ml acetylated BSA, 1 mM DTT, 20 mM Tris–HCl (pH 7.9) and 2–3 ng of DNA substrate in 25 μl of reaction volume. For TFIIH, a 3'–5' XPB-specific substrate was used. This substrate was generated from a 32P-labeled 19 nt long complementary oligonucleotide annealed to a 49 nt long oligonucleotide, resulting in a 5' blunt end (with respect to the longer oligonucleotide) partial duplex DNA. The reactions were stopped and run on 12% PAGE in 1x TBE.
ATPase assays were as described previously (19,22). The conditions were similar to the helicase assay above, except that 1 μM of ATP was added instead of 4 mM ATP, reaction volume was 12.5 μl, and the reaction times reduced to 20 min. For the XPB and TFIIH ATPase assay, 10 ng of single-stranded DNA and 50 ng of plasmid DNA were added, respectively, unless otherwise specified. The reactions were stopped and run on PEI-TLC plates in 0.75 M potassium phosphate (pH 4.0). All signals were detected by PhosphorImager and analyzed by ImageQuant 5.2 software.
Co-immunoprecipitation (CoIP) and template pull-down assays
An aliquot of 200 μl of bacterial lysate (2 μg total protein/μl) containing expressed TFIIE or its N-terminal truncation mutants was incubated with 200 μl of bacterial lysate (2 μg total protein/μl) containing expressed TFIIE? for 45 min at 4°C. An aliquot of 20 μl of Ni-NTA resin was added to the mixture to bind TFIIE? for an additional 45 min. Resins were washed twice with 400 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl and eluted with 2x SDS buffer . The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-FLAG M2 peroxidase conjugate (Sigma).
For TFIIE and TFIIE? CoIP, 130 μg of FLAG tagged TFIIE non-expressed or expressed bacterial lysate were mixed with 25 μl of anti-FLAG resin for 45 min at 4°C. Resin was washed as above. An aliquot of 200 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl and 1 μg of purified TFIIE? or its mutants were added to the spun down resin and further mixed for 45 min at 4°C. Resins were washed and eluted with 2x SDS buffer. The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-TFIIE? (Santa Cruz Biotechnology). For FLAG-tagged TFIIE? and His6 tagged TFIIE? CoIP, Anti-His peroxidase conjugate (Qiagen) was used in western blot detection.
For XPB and TFIIE? CoIP, 8 μg of GST or XPB were pre-incubated with 2 μg of TFIIE? or its mutants on ice for 45 min. Subsequently, 50 μl of glutathione resin (Amersham) and 400 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl were mixed with the pre-incubated proteins for 45 min at 4°C. Resins were washed and eluted with 2x SDS buffer. The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-TFIIE? (Santa Cruz Biotechnology).
The pre-initiation complex pull-down assay was performed similarly as described previously (42). Briefly, the 100 μl reaction contained 400 μg of TFIIE? depleted extract, 7.5 mM MgCl2, 0.05% NP-40, 1 mg/ml of acetylated bovine albumin, 1 μg of pGEM, 20 ng of TFIIE? or its mutants, 15 ng of TFIIE, 250 U Gal4-AH (ProteinOne) and the magnetic beads (Dynal) with the biotinylated G9E4 or promoter-less DNA (320 nt long). An aliquot of 0.2 pmol of E4 fragment was also added to each reaction. Incubations were at room temperature for 30 min. The beads were washed twice with 100 μl of Buffer D with 0.5% of NP-40.
In vitro transcription
HeLa nuclear extract (43) was immunodepleted with TFIIE using Protein A-agarose pre-bound with anti-IIE beta antibody (IIE? C-21 from Santa Cruz Biotechnology). The immunoprecipitation protocol was as described previously (44). In vitro transcription was performed as described previously (43). Briefly, the 25 μl reaction contained 70 μg of immunodepleted extract, 4 mM MgCl2, 200 ng of linearized AdML fragment with 5 Gal sites and a G-less cassette or 400 ng of linearized E4 fragment with 9 Gal sites, 200 ng of Gal4-VP16 (Protein One), 0.4 mM of ATP, 0.4 mM of CTP and 0.016 mM of UTP (Perkin-Elmer) (and 0.4 mM of GTP was included for E4 transcription). An aliquot of 90 ng of IIE and 60 ng of wild-type IIE? or its mutants were allowed to incubate for 10 min before the addition of other components in the reaction. The reaction was stopped after 60 min at 30°C, phenol–chloroform extracted and ethanol precipitated. Formamide dye was added to the pellet, loaded on to a 6% urea polyacrylamide gel and run for 80 min at 32 W. The signals were detected by PhosphorImager and analyzed by ImageQuant 5.2 software.
RESULTS
TFIIE? stimulates XPB helicase and ATPase activities
We first tested the effect of the isolated TFIIE? subunit on the enzymatic activities of the isolated XPB subunit of TFIIH. The results of helicase assays using purified human XPB and TFIIE? are shown in the autoradiogram of Figure 1. Electrophoresis was used to follow the release of a radioactively labeled oligonucleotide (of length 35 nt) from a partial DNA duplex. The partial duplex is shown in lane 1, and the labeled oligonucleotide, released artificially by heat, is shown in lane 2. The lack of release in lane 3 confirms that TFIIE? has low intrinsic helicase activity. When XPB is assayed (lane 4), the known weak helicase activity is represented by the low amount of released substrate. When TFIIE? is added to XPB, there is more released substrate (lanes 5 and 6) showing that TFIIE? has stimulated the XPB helicase activity. The stoichiometries of TFIIE? and XPB are 1:2 in lane 5 and 1:1 in lane 6. In Figure 1C, the stimulation of XPB helicase by TFIIE? is confirmed and shown to be unrelated to the GST tag attached to the recombinant protein.
Figure 1 TFIIE? stimulates XPB helicase activity. (A) Helicase assay. Lane 1, substrate alone; lane 2, substrate heated for 2 min to unanneal radioactively labeled oligonucleotide; lane 3, 0.8 μg TFIIE? was added; lane 4, 2.4 μg of XPB was added; lanes 5 and 6, 0.4 and 0.8 μg of TFIIE were added to 2.4 μg of XPB, respectively. (B) A graphical representation of lane 4–6 in (A). (C) Helicase assay. Lane 1, substrate alone; lane 2, 0.8 μg of TFIIE? and 2.4 μg of GST were added; lane 3, 0.8 μg of TFIIE? and 11 μg of GST were added; lane 4, 0.8 μg of TFIIE? were added to 2.4 μg of XPB. (D) ATPase assay. Lane 1, ATP alone; lane 2, 0.13 μg of TFIIE? was added; lane 3, 0.8 μg of XPB was added; lane 4 contained both 0.13 μg of TFIIE? and 0.8 μg of XPB. The ATPase stimulation was 1.8 ± 0.4-fold.
A titration was used to follow the quantitative stimulation (Figure 1B), which increases with increasing amounts of TFIIE?. When the amount is raised further, the helicase activity falls (data not shown). This may be related to the prevalence of beta dimers at the higher concentration, as TFIIE? exists in monomeric and dimeric states (11). At the optimal TFIIE? concentration the stimulation of XPB helicase activity is 4- to 5-fold under these conditions.
The effect of the isolated beta subunit on the XPB ATPase activity in the presence of DNA was also tested. This activity is needed for helicase action but also may act independently in some circumstances (33,45). The ATPase is assayed by observing the release of radioactive phosphate from gamma-labeled ATP, and Figure 1D shows that XPB (lane 3) but not TFIIE? (lane 2) has ATPase activity, as expected. When TFIIE? is added to XPB, its ATPase activity increases 2-fold (lane 4). We conclude that TFIIE? can stimulate the helicase and ATPase activities of the isolated XPB subunit of TFIIH.
Domain determinants of helicase stimulation
We used a series of C-terminal deletion mutants (Figure 2A) to learn the region(s) of TFIIE? that is important for the XPB helicase stimulation. C-terminal deletions were used because the small C-terminal basic region–helix–loop sequence is crowded with potential functions and is needed for transcription (36,38). When this small 35 amino acid region is removed, there is much less stimulation (Figure 2B, lane 5 versus lane 4). The residual helicase stimulation is removed upon further deletion, which also includes the leucine-rich sigma 3 region (lane 7 versus lane 3; summarized in Figure 2C). It appears that a large region of the C-terminus is involved in stimulation.
Figure 2 The C-terminus of TFIIE? is important for the stimulation of XPB helicase activity. (A) A diagram of TFIIE? functional domains (36) and the C-terminal truncation mutants of IIE? were depicted. (B) Helicase assay, conditions as in Figure 1A. Lane 1, substrate alone; lane 2, substrate heated for 2 min; lane 3, XPB was added; lane 4, as in lane 3, except wild-type TFIIE? was added; lane 5, equimolar of 257–291 as in lane 4 TFIIE? was added to XPB; lane 6, 197–291 was added to XPB; lane 7, 149–291 was added to XPB. (C) A graphical representation of XPB helicase stimulation by wild-type and various TFIIE? mutants.
XPB is in a different context when incorporated into the multi-subunit TFIIH, which contains nine other polypeptides, including its binding partner p52 and another helicase, XPD. We wished to learn whether TFIIE? would still stimulate XPB helicase activity in this context. To avoid a signal from the XPD helicase activity, an XPB-specific 3'–5' helicase substrate was used (21,22).
Figure 3A shows that the isolated beta subunits retain the ability to stimulate the XPB helicase in the context of intact TFIIH (compare lane 8 with lane 7 and compare lane 6 with lane 5), although the stimulation is less using this assay (2-fold). Figure 3B shows that TFIIE? also stimulates the ATPase activity of TFIIH (lane 4 versus lane 3), as would be expected based on the property of isolated XPB (see above). We conclude that TFIIE? stimulates the enzymatic activities of XPB, both in isolated form and in the context of TFIIH.
Figure 3 TFIIE? stimulates TFIIH helicase and ATPase activity. (A) Helicase assay. Lane 1, XPB 3'–5' helicase specific substrate; lane 2, substrate was heated for 1 min; lane 3, 5 and 7 contained 30 ng of TFIIH and the reaction times were 15, 30 and 45 min, respectively; lane 4, 6 and 8 contained 80 ng of TFIIE? and 30 ng of TFIIH and the reaction times are 15, 30 and 45 min, respectively. (B) ATPase assay. Lane 1, ATP alone; lane 2, 80 ng of TFIIE? was added; lane 3, 60 ng of TFIIH was added; lane 4 contained 80 ng of TFIIE? and 60 ng of TFIIH. The ATPase stimulation was 2.2 ± 0.4-fold.
TFIIE counteracts TFIIE? stimulation of the XPB helicase
In contrast to the above results, the TFIIE complex, consisting of an 2?2 tetramer, is known to have the capacity to negatively regulate the helicase activity of XPB and TFIIH (14,39). This negative regulation is lessened in the presence of an antibody to the alpha subunit (39). These observations raise the possibility that the beta subunit has stimulation potential but in the context of intact TFIIE it can be moderated by the inhibitory potential of the alpha subunit. To test directly whether alpha can inhibit the stimulatory effect of beta, TFIIE was allowed to form a complex with TFIIE? before the addition of XPB and its helicase substrate. As alpha is titrated against beta, the stimulatory activity of beta falls progressively (Figure 4A, lanes 3–5 and summarized in Figure 4B), confirming the moderation effect of the alpha subunit of the helicase stimulation by the beta subunit.
Figure 4 TFIIE prevents TFIIE? from stimulating XPB. (A) Helicase assay. Lane 1, substrate alone; lane 2, XPB was added; lane 3, 0.8 μg of TFIIE? and XPB were included. For lanes 4 and 5, 1.6 and 3.2 μg, respectively, of IIE were incubated with 0.8 μg of TFIIE? for 15 min before the addition of XPB and the substrate. (B) A graphical representation of the helicase activities of samples in lanes 3–5.
TFIIE inhibits the XPB helicase directly, rather than working through TFIIE?
We tested the possibility that TFIIE moderates the TFIIE? helicase stimulation through its interaction with TFIIE?. Two N-terminal deletion mutants of TFIIE were constructed (Figure 5A) that would be expected to prevent the formation of a complex with TFIIE? (16). The CoIP experiment in Figure 5B confirms that the wild-type TFIIE binds TFIIE? (lane 2) but that N-terminal truncations of amino acids 1–49 (lane 3) or 1–66 (lane 4) prevent the association of TFIIE with TFIIE?.
Figure 5 TFIIE binds to XPB to counteract TFIIE? stimulation. (A) Western blot of wild-type TFIIE and its mutants. (B) CoIP of TFIIE? with wild-type TFIIE and its mutants. (C) Helicase assay. Lane 3, XPB was added; lane 4, TFIIE? and XPB were added (70% of substrate was unwound); lanes 5 and 6 as in lane 4, except 2.4 μg of TFIIE or 50–439 were added, respectively (30% of substrate were unwound for both).
The non-interacting mutant 50–439 was then tested for the ability to inhibit TFIIE?-stimulated XPB helicase activity. Figure 5C shows that the inhibition is still intact, even though the mutant TFIIE can no longer bind TFIIE?. The inhibition by the non-interacting alpha mutant is comparable with that of wild-type TFIIE (compare lanes 5 and 6 with the stimulated control in lane 4). We conclude that the ability of TFIIE to inhibit stimulation of XPB helicase activity does not depend on its association with TFIIE?. Apparently, TFIIE? and TFIIE can act independently directly on XPB, one to stimulate and the other to inhibit.
TFIIE? mutants defective in the stimulation of the XPB helicase are also defective in transcription
To examine the physiological relevance of the IIE beta stimulation of the XPB helicase, we attempted to identify and test transcription of IIE beta mutants that would be specifically defective in helicase stimulation. The data of Figure 2 showed that the deletion of the C-terminal BR-HL region lessens helicase stimulation and further deletion through the LR sigma 3 region reduces the residual activity, implicating multiple regions. The C-terminal BR-HLH is dense with overlapping functions (38). These include the IIE–alpha interaction (36,38) that is presumed to be important for recruitment. Therefore, mutants in this region would be expected to alter multiple interactions, not just stimulation of the XPB helicase. In contrast, no functions have yet been ascribed to the LR sigma 3 region, although it is known that its deletion results in a loss of basal transcription activity on a linearized template (36).
Several mutants were made in this region. These changed highly conserved hydrophobic residues to the hydrophilic serine (I171S and I189S). The extract was immunodepleted so as to make transcription depend on added wild-type TFIIE (44); this was reproduced with wild-type TFIIE (Figure 6A, depleted lane 1 versus lane 2 with TFIIE added back). In contrast, transcription was strongly (Figure 6A, lane 3 versus lane 2) or partially (lane 4 versus lane 2) defective when the mutant forms of TFIIE were added back. These two mutants were also found to be associated with defects in stimulated XPB helicase activity (Figure 6B, lanes 3 and 4 versus the wild-type in lane 2). Defects in XPB helicase activity are known to be led to defects in transcription (5,28), so the transcription defect associated with these TFIIE mutants is most likely due to lower XPB helicase activity (Figure 6A and B), although other contributions cannot be excluded.
Figure 6 Mutants that fail to stimulate XPB helicase activity also have transcription defects. (A) In vitro transcription at the AdML promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIE? and TFIIE; lane 3, with mutant I171S and TFIIE (0% transcription activity compared with wild-type); lane 4, with mutant I189S and TFIIE (60% transcription activity compared with wild-type). (B) Helicase assay. Lane 1, 2 μg of XPB; lane 2, XPB and 1.2 μg of wild-type TFIIE? (3-fold stimulation); lane 3, XPB and 1.2 μg of I171S; lane 4, XPB and 1.2 μg of I189S. Only the released oligo is shown. (C) CoIP with TFIIE? and general transcription factors. Top panel: lane 1, anti-FLAG resin plus wild-type TFIIE?; lanes 2–4 anti-FLAG resin pre-bound with FLAG-tagged TFIIE plus wild-type TFIIE or its mutants. Middle panel: lane 1, glutathione resin with GST plus wild-type TFIIE?; lanes 2–4 glutathione resin with GST–XPB plus wild-type TFIIE? or its mutants. Bottom panel: lane 1, anti-FLAG resin plus wild-type TFIIE? with His6 tag; lanes 2–4, anti-FLAG resin pre-bound with FLAG-tagged wild type TFIIE? 6x His tag or its mutants. (D) Pre-initiation complex formation of wild-type TFIIE? and its mutants. Lane 1, beads with equimolar of the template without the E4 promoter and gal sites are incubated with TFIIE? depleted extract complement with recombinant wild-type TFIIE. Lanes 2–4, beads bound to G9E4 template are incubated with recombinant wild-type TFIIE, I171S or I189S, respectively. The left panel indicates the transcription factors that are being probed. (E) In vitro transcription at the G9E4 promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIE; lane 3, with mutant I171S and TFIIE; lane 4, with mutant I189S and TFIIE.
Several other in vitro functions were found to be normal using a CoIP assay (8). First, the two beta mutants bind TFIIE comparably with wild-type (Figure 6C, top row, compare mutants in lanes 3 and 4 with the wild-type in lane 2). Second, the beta subunits are known to dimerize as part of intact TFIIE (11) and this beta dimerization function is normal (Figure 6C, bottom panel). Third, the binding of beta to XPB is also comparable for the mutants and wild-type (Figure 6C, middle panel).
Although this region would not be expected to affect pre-initiation complex formation, as the C-terminus binds the alpha subunit presumably required for assembly, this was tested directly using an immobilized template pull-down assay with a biotinylated promoter (42). A G9E4 template was chosen over the AdML G-less cassette template used earlier because the G-less cassette appeared to bind a significant amount of factors non-specifically (data not shown). The wild-type transcription complex was shown to be functional as hydrolysable NTPs resulted in polymerase II and TFIIH leaving the complex (data not shown) (42).
Figure 6D shows that transcription factors were recruited to the template, whether the wild-type or the mutant forms of beta were present. These include the RNA polymerase itself (second to bottom row) and two subunits of TFIIH, XPB (top row) and p62 (bottom row); the control in this case was the use of promoter-less DNA (compare this control in column 1 with promoter DNA pull-downs in columns 2–4). Although the wild-type and mutant TFIIE subunits were also pulled down comparably, we could not confirm the promoter specificity of the interactions (again compare with column 1 in this context).
Overall, the data demonstrate that the TFIIE? mutants recruit TFIIH to the template but are defective in stimulating the TFIIH XPB helicase activity. The associated transcription defect is re-confirmed using the same E4 template used in the template pull-down assay (lanes 3 and 4 versus lane 2 of Figure 6E), which complements the transcription data for the ML template (Figure 6A).
DISCUSSION
The ATP-dependent XPB helicase activity of TFIIH is used in triggering promoter-bound RNA polymerase II to progress through initiation and into early elongation (27,28,30,31,34). The enzyme activity in vitro is known to be very low; contributions may include the lack of a binding partner, such as p44, which stimulates the TFIIH XPD helicase (25,26) and inhibition by the alpha subunit of TFIIE (39). In this paper, we show that the ATPase and helicase activities of XPB can be strengthened by its partnering with the beta subunit of TFIIE. This stimulation appears to be functionally significant as point mutations in TFIIE? that have defects in XPB stimulation are also defective in transcription; in contrast, the mutations do not interfere with the recruitment of XPB to the transcription complex or with TFIIE? binding to TFIIE.
The stimulatory activity of the TFIIE? subunit can be counter-acted by the inhibitory activity of its alpha partner subunit. The current data show that the stimulatory activity of TFIIE? and the inhibitory activity of TFIIE act independently, i.e. each works directly on XPB. This suggests that the TFIIE complex may be built so that it has the potential to either activate or inhibit. Photo-cross-linking data (46) suggest that ATP causes TFIIE to re-arrange so as to better engage the downstream region of the transcription bubble (from +4 to +8) during promoter opening, while no accompanying change in the positioning of the beta subunit was detected. It is possible that this re-arrangement within TFIIE releases the beta subunit to stimulate the ATP-dependent XPB helicase to enhance the subsequent promoter escape steps. The data show that it is the C-terminus of TFIIE? that accomplishes this. Because this region interacts with many components of the transcription complex (36,38), the data raise the possibility that it has a role in multiple re-modeling interactions subsequent to complex assembly (47–49).
Among RNA polymerases, only RNA polymerase II requires a helicase to transit to elongation mode following recruitment to the promoter (50–52), and TFIIH and TFIIE have evolved to meet this unique requirement. This mechanism enhances the potential diversity of regulation and factors may exist that target the TFIIE? helicase stimulatory activity to control the entry of the promoter-bound RNA polymerase into elongation phase. This transition involves the opening of DNA around the start site (7,52) and the release of factors holding RNA polymerase to the promoter (48,49), leading to the addition of factors that couple transcription elongation and RNA processing (47). These steps cannot occur without the action of the ATP-dependent XPB helicase (13,30,31,36,38), which the current data suggest that it is controlled by TFIIE?. Such considerations raise the possibility that any or all steps could be potential targets for regulation via the TFIIE?-stimulated ATP-dependent helicase activity, although the recent demonstration that helicase action comes into play directly after promoter opening (Y. C. Lin and J. D. Gralla, accepted by Nature Structural and Molecular Biology) favors a primary role in providing the energy needed to re-model open pre-initiation complexes and drive RNA polymerase into elongation mode.
ACKNOWLEDGEMENTS
The authors thank David Vargas for purifying some of the proteins and for technical assistance. The authors are grateful to Hao Duong (UCLA, Department of Chemistry and Biochemistry) for providing TFIIE and TFIIE? during the initial phase of these studies, Dr Jean-Marc Egly for the XPB clone and Dr Mike Carey for materials and protocols for immobilized template assay. This research was supported by USPHS grant NIHGM49048. Funding to pay the Open Access publication charges for this article was provided by USPHS.
REFERENCES
Hampsey, M. (1998) Molecular genetics of the RNA polymerase II general transcriptional machinery Microbiol. Mol. Biol. Rev., 62, 465–503 .
Orphanides, G., Lagrange, T., Reinberg, D. (1996) The general transcription factors of RNA polymerase II Genes Dev., 10, 2657–2683 .
Lee, T.I. and Young, R.A. (2000) Transcription of eukaryotic protein-coding genes Annu. Rev. Genet., 34, 77–137 .
Buratowski, S., Hahn, S., Guarente, L., Sharp, P.A. (1989) Five intermediate complexes in transcription initiation by RNA polymerase II Cell, 56, 549–561 .
Dvir, A., Conaway, J.W., Conaway, R.C. (2001) Mechanism of transcription initiation and promoter escape by RNA polymerase II Curr. Opin. Genet. Dev., 11, 209–214 .
Holstege, F.C., Fiedler, U., Timmers, H.T. (1997) Three transitions in the RNA polymerase II transcription complex during initiation EMBO J., 16, 7468–7480 .
Holstege, F.C., van der Vliet, P.C., Timmers, H.T. (1996) Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH EMBO J., 15, 1666–1677 .
Flores, O., Lu, H., Reinberg, D. (1992) Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH J. Biol. Chem., 267, 2786–2793 .
Wolner, B.S. and Gralla, J.D. (1997) Promoter activation via a cyclic AMP response element in vitro J. Biol. Chem., 272, 32301–32307 .
Spangler, L., Wang, X., Conaway, J.W., Conaway, R.C., Dvir, A. (2001) TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA Proc. Natl Acad. Sci. USA, 98, 5544–5549 .
Peterson, M.G., Inostroza, J., Maxon, M.E., Flores, O., Admon, A., Reinberg, D., Tjian, R. (1991) Structure and functional properties of human general transcription factor IIE Nature, 354, 369–373 .
Park, E., Guzder, S.N., Koken, M.H., Jaspers-Dekker, I., Weeda, G., Hoeijmakers, J.H., Prakash, S., Prakash, L. (1992) RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability Proc. Natl Acad. Sci. USA, 89, 11416–11420 .
Goodrich, J.A. and Tjian, R. (1994) Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II Cell, 77, 145–156 .
Inostroza, J., Flores, O., Reinberg, D. (1991) Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of general transcription factor IIE J. Biol. Chem., 266, 9304–9308 .
Ohkuma, Y., Sumimoto, H., Horikoshi, M., Roeder, R.G. (1990) Factors involved in specific transcription by mammalian RNA polymerase II: purification and characterization of general transcription factor TFIIE Proc. Natl Acad. Sci. USA, 87, 9163–9167 .
Ohkuma, Y., Hashimoto, S., Wang, C.K., Horikoshi, M., Roeder, R.G. (1995) Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure–function studies of TFIIE-alpha Mol. Cell. Biol., 15, 4856–4866 .
Keriel, A., Stary, A., Sarasin, A., Rochette-Egly, C., Egly, J.M. (2002) XPD mutations prevent TFIIH-dependent transactivation by nuclear receptors and phosphorylation of RARalpha Cell, 109, 125–135 .
Schultz, P., Fribourg, S., Poterszman, A., Mallouh, V., Moras, D., Egly, J.M. (2000) Molecular structure of human TFIIH Cell, 102, 599–607 .
Ohkuma, Y. and Roeder, R.G. (1994) Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation Nature, 368, 160–163 .
Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J.H., Chambon, P., Egly, J.M. (1993) DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor Science, 260, 58–63 .
Schaeffer, L., Moncollin, V., Roy, R., Staub, A., Mezzina, M., Sarasin, A., Weeda, G., Hoeijmakers, J.H., Egly, J.M. (1994) The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor EMBO J., 13, 2388–2392 .
Roy, R., Schaeffer, L., Humbert, S., Vermeulen, W., Weeda, G., Egly, J.M. (1994) The DNA-dependent ATPase activity associated with the class II basic transcription factor BTF2/TFIIH J. Biol. Chem., 269, 9826–9832 .
Svejstrup, J.Q., Vichi, P., Egly, J.M. (1996) The multiple roles of transcription/repair factor TFIIH Trends Biochem. Sci., 21, 346–350 .
Serizawa, H., Conaway, J.W., Conaway, R.C. (1994) An oligomeric form of the large subunit of transcription factor (TF) IIE activates phosphorylation of the RNA polymerase II carboxyl-terminal domain by TFIIH J. Biol. Chem., 269, 20750–20756 .
Coin, F., Marinoni, J.C., Rodolfo, C., Fribourg, S., Pedrini, A.M., Egly, J.M. (1998) Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH Nature Genet., 20, 184–188 .
Dubaele, S., Proietti De Santis, L., Bienstock, R.J., Keriel, A., Stefanini, M., Van Houten, B., Egly, J.M. (2003) Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients Mol. Cell, 11, 1635–1646 .
Hwang, J.R., Moncollin, V., Vermeulen, W., Seroz, T., van Vuuren, H., Hoeijmakers, J.H., Egly, J.M. (1996) A 3'5' XPB helicase defect in repair/transcription factor TFIIH of xeroderma pigmentosum group B affects both DNA repair and transcription J. Biol. Chem., 271, 15898–15904 .
Tirode, F., Busso, D., Coin, F., Egly, J.M. (1999) Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD and cdk7 Mol. Cell, 3, 87–95 .
Douziech, M., Coin, F., Chipoulet, J.M., Arai, Y., Ohkuma, Y., Egly, J.M., Coulombe, B. (2000) Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein–DNA photo-cross-linking Mol. Cell. Biol., 20, 8168–8177 .
Fukuda, A., Nogi, Y., Hisatake, K. (2002) The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation Proc. Natl Acad. Sci. USA, 99, 1206–1211 .
Moreland, R.J., Tirode, F., Yan, Q., Conaway, J.W., Egly, J.M., Conaway, R.C. (1999) A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II J. Biol. Chem., 274, 22127–22130 .
Jawhari, A., Laine, J.P., Dubaele, S., Lamour, V., Poterszman, A., Coin, F., Moras, D., Egly, J.M. (2002) p52 Mediates XPB function within the transcription/repair factor TFIIH J. Biol. Chem., 277, 31761–31767 .
Winkler, G.S., Sugasawa, K., Eker, A.P., de Laat, W.L., Hoeijmakers, J.H. (2001) Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF Biochemistry, 40, 160–165 .
Bradsher, J., Coin, F., Egly, J.M. (2000) Distinct roles for the helicases of TFIIH in transcript initiation and promoter escape J. Biol. Chem., 275, 2532–2538 .
Maxon, M.E., Goodrich, J.A., Tjian, R. (1994) Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance Genes Dev., 8, 515–524 .
Okamoto, T., Yamamoto, S., Watanabe, Y., Ohta, T., Hanaoka, F., Roeder, R.G., Ohkuma, Y. (1998) Analysis of the role of TFIIE in transcriptional regulation through structure–function studies of the TFIIE? subunit J. Biol. Chem., 273, 19866–19876 .
Chen, J., Larochelle, S., Li, X., Suter, B. (2003) Xpd/Ercc2 regulates CAK activity and mitotic progression Nature, 424, 228–232 .
Watanabe, T., Hayashi, K., Tanaka, A., Furumoto, T., Hanaoka, F., Ohkuma, Y. (2003) The carboxy terminus of the small subunit of TFIIE regulates the transition from transcription initiation to elongation by RNA polymerase II Mol. Cell. Biol., 23, 2914–2926 .
Drapkin, R., Reardon, J.T., Ansari, A., Huang, J.C., Zawel, L., Ahn, K., Sancar, A., Reinberg, D. (1994) Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II Nature, 368, 769–772 .
Yan, M. and Gralla, J.D. (1997) Multiple ATP-dependent steps in RNA polymerase II promoter melting and initiation EMBO J., 16, 7457–7467 .
van Vuuren, A.J., Vermeulen, W., Ma, L., Weeda, G., Appeldoorn, E., Jaspers, N.G., van der Eb, A.J., Bootsma, D., Hoeijmakers, J.H., Humbert, S., et al. (1994) Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH) EMBO J., 13, 1645–1653 .
Johnson, K.M., Wang, J., Smallwood, A., Carey, M. (2004) The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly Methods Enzymol., 380, 207–219 .
Dignam, J.D., Lebovitz, R.M., Roeder, R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei Nucleic Acids Res., 11, 1475–1489 .
Park, C.H., Mu, D., Reardon, J.T., Sancar, A. (1995) The general transcription-repair factor TFIIH is recruited to the excision repair complex by the XPA protein independent of the TFIIE transcription factor J. Biol. Chem., 270, 4896–4902 .
Evans, E., Moggs, J.G., Hwang, J.R., Egly, J.M., Wood, R.D. (1997) Mechanism of open complex and dual incision formation by human nucleotide excision repair factors EMBO J., 16, 6559–6573 .
Kim, T.K., Ebright, R.H., Reinberg, D. (2000) Mechanism of ATP-dependent promoter melting by transcription factor IIH Science, 288, 1418–1422 .
Svejstrup, J.Q. (2004) The RNA polymerase II transcription cycle: cycling through chromatin Biochim. Biophys. Acta, 1677, 64–73 .
Yudkovsky, N., Ranish, J.A., Hahn, S. (2000) A transcription reinitiation intermediate that is stabilized by activator Nature, 408, 225–229 .
Yean, D. and Gralla, J.D. (1999) Transcription reinitiation rate: a potential role for TATA box stabilization of the TFIID:TFIIA:DNA complex Nucleic Acids Res., 27, 831–838 .
Paule, M.R. and White, R.J. (2000) Survey and summary: transcription by RNA polymerases I and III Nucleic Acids Res., 28, 1283–1298 .
Geiduschek, E.P. and Kassavetis, G.A. (2001) The RNA polymerase III transcription apparatus J. Mol. Biol., 310, 1–26 .
Wang, W., Gralla, J.D., Carey, M. (1992) The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA Genes Dev., 6, 1716–1727 .(Yin C. Lin and Jay D. Gralla*)
*To whom correspondence should be addressed. Tel: +1 310 825 1620; Fax: +1 310 267 2302; Email: gralla@chem.ucla.edu
ABSTRACT
TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.
INTRODUCTION
Eukaryotic class II transcription requires at least 30 polypeptides for basal transcription and many more are needed for properly regulated transcription. These polypeptides include RNA polymerase II and components of the general transcription factors. TFIID and TFIIB recruit the polymerase and TFIIF to the promoter, and other factors modify the transcription complex to allow it to proceed into early elongation phase (1–3). In the step-wise model of assembly, TFIIE and TFIIH are added immediately after polymerase (4) and are used in the opening of the DNA and in the initial steps of RNA synthesis, including promoter escape (5–10). TFIIE and TFIIH are essential proteins in vivo (11,12). In vitro, TFIIE and TFIIH are required for these transcription steps, although their function can be partially bypassed on some supercoiled templates (13).
TFIIE is composed of two subunits, a larger 56 kDa subunit TFIIE and a smaller 34 kDa subunit TFIIE? (14,15). The TFIIE heterotetramer (2?2) has no known catalytic activity and recruits TFIIH to complete the assembly pathway (4,8,16,17). TFIIH is a large complex with 10 subunits and several enzymatic activities, including ATP hydrolysis, energy-dependent DNA unwinding and phosphorylation (18–24). Two subunits of TFIIH, XPB and XPD, can accomplish DNA unwinding, although the polarity of the process is different for each (20–22). The XPD ATP-dependent helicase is used to open DNA during repair, and its activity within TFIIH is strong owing to the activation by its partner subunit p44 (25,26). The XPB ATP-dependent helicase is used to open the promoter DNA during transcription and is also used in promoter escape (27–31). Its helicase activity within TFIIH is weaker and its p52 partner subunit does not enhance this activity (32). XPB and XPD have other roles that appear to be independent of helicase action. The ATPase activity of XPB is needed for the activation of the XPG endonuclease during nucleotide excision repair (33). XPD provides structural integrity to TFIIH (25,26), especially to the CAK kinase complex that is involved in the transition from transcription initiation to elongation (16,19,24,26,28,34–36) and other cellular processes (17,37).
Although TFIIE has no catalytic activities (14,15), it can influence those of TFIIH (16,24,36,38). It has been reported to positively regulate the ATPase and kinase activities of TFIIH (19) and negatively regulate its helicase activity (39). The negative regulation is specific to the XPB helicase (39). From these observations, it has been suggested that TFIIE inhibits the ability of TFIIH to open promoter DNA until the complete complement of general transcription factors is assembled, at which time this constraint may be relieved. Overall, TFIIE and TFIIH likely work together to modify the pre-initiation complex so as to allow the RNA polymerase to function in transcription initiation and to transit to early elongation. During this critical transition process, the mRNA becomes capped and TFIIH begins to catalyze the staged series of changes in the RNA polymerase CTD that sets the stage for coupled elongation and RNA processing events. Although TFIIE is required for this transition (19,36), its role has not been clearly established. The role of the beta subunit is of particular interest, as its C-terminus is crowded with interaction domains for other general transcription factors (36,38).
Both the alpha and beta subunits of TFIIE can interact with XPB (35) and it has been suggested that the alpha subunit is dominant for the inhibition of the XPB function (39). No specific role has emerged for the interaction of the beta subunit with XPB, although the possibility has been raised that it functions in the transition into early elongation phase (36,38). Because promoter opening and escape require the XPB ATPase and helicase activities (5,28,31,40), we have investigated the possible role of the TFIIE beta subunit in modulating these activities. Below, we show that the isolated beta subunit can stimulate the XPB helicase and ATPase activities and that such stimulation has a functional role in transcription.
MATERIALS AND METHODS
Protein expression and purification
The human XPB (ERCC3) clone was a gift from the J. -M Egly laboratory. Glutathione S-transferase (GST)-tagged XPB was expressed and partially purified as described previously (22,41) with the following modifications. An aliquot of 400 ml of Escherichia coli cells were induced with 0.1 mM of isopropyl-?-D-thiogalactopyranoside (IPTG) for 30 min at 37°C. The cell pellets were collected and re-suspended in 10 ml of 1x phosphate-buffered saline (PBS) with 1% TritonTM X-100. An aliquot of 150 μl of Glutathione Sepharose 4B was used in the batch purification protocol as described previously (Amersham Pharmacia Biotech). Each wash used 10 ml of 1x PBS with 1% TritonTM X-100 for a total of four washes. The XPB was eluted with 50 mM Tris–HCl (pH 7.9), 10 mM reduced glutathione and 50% glycerol. The eluted sample was dialyzed against 50 mM Tris–HCl (pH 7.9) and 50% glycerol to remove the reduced glutathione. GST was expressed and purified the same way.
Human TFIIE and TFIIE? clones were obtained from Dr K. Yokomori. Human TFIIE?, its C-terminal truncated mutants (stop codons were placed accordingly using QuikChangeTM site-directed mutagenesis kit from Stratagene) and point mutants with His6 tagged were expressed in 4–500 ml of E.coli BL21(DE3) cells in 0.3 mM IPTG, 2% glucose and Luria–Bertani for 1 h at 37°C after OD600 nm reached 0.5. Ni-based resin (Qiagen and Active Motive) was used for purification (see Qiagen's manual for purification procedures and solutions). Proteins were analyzed on 12% Tris/Glycine SDS–PAGE with the correct molecular weights and were >95% pure. The proteins contained low but detectable helicase activity.
Human TFIIE with a His6 tag was a gift from Hao Duong (UCLA, Department of Chemistry and Biochemistry). Human TFIIE and N-terminal truncated mutants with an N-terminal FLAG tag were cloned into pET11a (Novagen). They were expressed the same way as TFIIE? (see above) and were purified using ANTI-FLAG? M2-Agarose (Sigma, see the manufacturer's purification procedures and solutions). The proteins were eluted with FLAG peptide. They were analyzed on 10% Tris/Glycine SDS–PAGE and were >95% pure.
Human TFIIH was purchased from Protein One (http://www.proteinone.com).
Helicase and ATPase assays
Helicase assays were as described previously (21,22,39). Briefly, 20 ng of 32P-labeled oligonucleotide (5'-AAACGACGGCCAGTGAATTCGAGCTCGGTACCCGG-3') complementary to M13mp19+ was annealed to 1 μg of the single-stranded plasmid DNA in 25 mM NaCl and 2.5 mM MgCl2. The duplex substrate was then purified in a Sephadex 200 size exclusion column. An aliquot of 400–500 ng of duplex was recovered free of un-annealed labeled oligonucleotide. Helicase assays for XPB and TFIIE? or its mutants were incubated for 30 min at 37°C in the presence of 4 mM MgCl2, 4 mM ATP, 50 μg/ml acetylated BSA, 1 mM DTT, 20 mM Tris–HCl (pH 7.9) and 2–3 ng of DNA substrate in 25 μl of reaction volume. For TFIIH, a 3'–5' XPB-specific substrate was used. This substrate was generated from a 32P-labeled 19 nt long complementary oligonucleotide annealed to a 49 nt long oligonucleotide, resulting in a 5' blunt end (with respect to the longer oligonucleotide) partial duplex DNA. The reactions were stopped and run on 12% PAGE in 1x TBE.
ATPase assays were as described previously (19,22). The conditions were similar to the helicase assay above, except that 1 μM of ATP was added instead of 4 mM ATP, reaction volume was 12.5 μl, and the reaction times reduced to 20 min. For the XPB and TFIIH ATPase assay, 10 ng of single-stranded DNA and 50 ng of plasmid DNA were added, respectively, unless otherwise specified. The reactions were stopped and run on PEI-TLC plates in 0.75 M potassium phosphate (pH 4.0). All signals were detected by PhosphorImager and analyzed by ImageQuant 5.2 software.
Co-immunoprecipitation (CoIP) and template pull-down assays
An aliquot of 200 μl of bacterial lysate (2 μg total protein/μl) containing expressed TFIIE or its N-terminal truncation mutants was incubated with 200 μl of bacterial lysate (2 μg total protein/μl) containing expressed TFIIE? for 45 min at 4°C. An aliquot of 20 μl of Ni-NTA resin was added to the mixture to bind TFIIE? for an additional 45 min. Resins were washed twice with 400 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl and eluted with 2x SDS buffer . The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-FLAG M2 peroxidase conjugate (Sigma).
For TFIIE and TFIIE? CoIP, 130 μg of FLAG tagged TFIIE non-expressed or expressed bacterial lysate were mixed with 25 μl of anti-FLAG resin for 45 min at 4°C. Resin was washed as above. An aliquot of 200 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl and 1 μg of purified TFIIE? or its mutants were added to the spun down resin and further mixed for 45 min at 4°C. Resins were washed and eluted with 2x SDS buffer. The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-TFIIE? (Santa Cruz Biotechnology). For FLAG-tagged TFIIE? and His6 tagged TFIIE? CoIP, Anti-His peroxidase conjugate (Qiagen) was used in western blot detection.
For XPB and TFIIE? CoIP, 8 μg of GST or XPB were pre-incubated with 2 μg of TFIIE? or its mutants on ice for 45 min. Subsequently, 50 μl of glutathione resin (Amersham) and 400 μl of 20 mM Tris–HCl (pH 7.9) and 100 mM NaCl were mixed with the pre-incubated proteins for 45 min at 4°C. Resins were washed and eluted with 2x SDS buffer. The samples were resolved by 10% Tris/Glycine SDS–PAGE and detected through western blot using Anti-TFIIE? (Santa Cruz Biotechnology).
The pre-initiation complex pull-down assay was performed similarly as described previously (42). Briefly, the 100 μl reaction contained 400 μg of TFIIE? depleted extract, 7.5 mM MgCl2, 0.05% NP-40, 1 mg/ml of acetylated bovine albumin, 1 μg of pGEM, 20 ng of TFIIE? or its mutants, 15 ng of TFIIE, 250 U Gal4-AH (ProteinOne) and the magnetic beads (Dynal) with the biotinylated G9E4 or promoter-less DNA (320 nt long). An aliquot of 0.2 pmol of E4 fragment was also added to each reaction. Incubations were at room temperature for 30 min. The beads were washed twice with 100 μl of Buffer D with 0.5% of NP-40.
In vitro transcription
HeLa nuclear extract (43) was immunodepleted with TFIIE using Protein A-agarose pre-bound with anti-IIE beta antibody (IIE? C-21 from Santa Cruz Biotechnology). The immunoprecipitation protocol was as described previously (44). In vitro transcription was performed as described previously (43). Briefly, the 25 μl reaction contained 70 μg of immunodepleted extract, 4 mM MgCl2, 200 ng of linearized AdML fragment with 5 Gal sites and a G-less cassette or 400 ng of linearized E4 fragment with 9 Gal sites, 200 ng of Gal4-VP16 (Protein One), 0.4 mM of ATP, 0.4 mM of CTP and 0.016 mM of UTP (Perkin-Elmer) (and 0.4 mM of GTP was included for E4 transcription). An aliquot of 90 ng of IIE and 60 ng of wild-type IIE? or its mutants were allowed to incubate for 10 min before the addition of other components in the reaction. The reaction was stopped after 60 min at 30°C, phenol–chloroform extracted and ethanol precipitated. Formamide dye was added to the pellet, loaded on to a 6% urea polyacrylamide gel and run for 80 min at 32 W. The signals were detected by PhosphorImager and analyzed by ImageQuant 5.2 software.
RESULTS
TFIIE? stimulates XPB helicase and ATPase activities
We first tested the effect of the isolated TFIIE? subunit on the enzymatic activities of the isolated XPB subunit of TFIIH. The results of helicase assays using purified human XPB and TFIIE? are shown in the autoradiogram of Figure 1. Electrophoresis was used to follow the release of a radioactively labeled oligonucleotide (of length 35 nt) from a partial DNA duplex. The partial duplex is shown in lane 1, and the labeled oligonucleotide, released artificially by heat, is shown in lane 2. The lack of release in lane 3 confirms that TFIIE? has low intrinsic helicase activity. When XPB is assayed (lane 4), the known weak helicase activity is represented by the low amount of released substrate. When TFIIE? is added to XPB, there is more released substrate (lanes 5 and 6) showing that TFIIE? has stimulated the XPB helicase activity. The stoichiometries of TFIIE? and XPB are 1:2 in lane 5 and 1:1 in lane 6. In Figure 1C, the stimulation of XPB helicase by TFIIE? is confirmed and shown to be unrelated to the GST tag attached to the recombinant protein.
Figure 1 TFIIE? stimulates XPB helicase activity. (A) Helicase assay. Lane 1, substrate alone; lane 2, substrate heated for 2 min to unanneal radioactively labeled oligonucleotide; lane 3, 0.8 μg TFIIE? was added; lane 4, 2.4 μg of XPB was added; lanes 5 and 6, 0.4 and 0.8 μg of TFIIE were added to 2.4 μg of XPB, respectively. (B) A graphical representation of lane 4–6 in (A). (C) Helicase assay. Lane 1, substrate alone; lane 2, 0.8 μg of TFIIE? and 2.4 μg of GST were added; lane 3, 0.8 μg of TFIIE? and 11 μg of GST were added; lane 4, 0.8 μg of TFIIE? were added to 2.4 μg of XPB. (D) ATPase assay. Lane 1, ATP alone; lane 2, 0.13 μg of TFIIE? was added; lane 3, 0.8 μg of XPB was added; lane 4 contained both 0.13 μg of TFIIE? and 0.8 μg of XPB. The ATPase stimulation was 1.8 ± 0.4-fold.
A titration was used to follow the quantitative stimulation (Figure 1B), which increases with increasing amounts of TFIIE?. When the amount is raised further, the helicase activity falls (data not shown). This may be related to the prevalence of beta dimers at the higher concentration, as TFIIE? exists in monomeric and dimeric states (11). At the optimal TFIIE? concentration the stimulation of XPB helicase activity is 4- to 5-fold under these conditions.
The effect of the isolated beta subunit on the XPB ATPase activity in the presence of DNA was also tested. This activity is needed for helicase action but also may act independently in some circumstances (33,45). The ATPase is assayed by observing the release of radioactive phosphate from gamma-labeled ATP, and Figure 1D shows that XPB (lane 3) but not TFIIE? (lane 2) has ATPase activity, as expected. When TFIIE? is added to XPB, its ATPase activity increases 2-fold (lane 4). We conclude that TFIIE? can stimulate the helicase and ATPase activities of the isolated XPB subunit of TFIIH.
Domain determinants of helicase stimulation
We used a series of C-terminal deletion mutants (Figure 2A) to learn the region(s) of TFIIE? that is important for the XPB helicase stimulation. C-terminal deletions were used because the small C-terminal basic region–helix–loop sequence is crowded with potential functions and is needed for transcription (36,38). When this small 35 amino acid region is removed, there is much less stimulation (Figure 2B, lane 5 versus lane 4). The residual helicase stimulation is removed upon further deletion, which also includes the leucine-rich sigma 3 region (lane 7 versus lane 3; summarized in Figure 2C). It appears that a large region of the C-terminus is involved in stimulation.
Figure 2 The C-terminus of TFIIE? is important for the stimulation of XPB helicase activity. (A) A diagram of TFIIE? functional domains (36) and the C-terminal truncation mutants of IIE? were depicted. (B) Helicase assay, conditions as in Figure 1A. Lane 1, substrate alone; lane 2, substrate heated for 2 min; lane 3, XPB was added; lane 4, as in lane 3, except wild-type TFIIE? was added; lane 5, equimolar of 257–291 as in lane 4 TFIIE? was added to XPB; lane 6, 197–291 was added to XPB; lane 7, 149–291 was added to XPB. (C) A graphical representation of XPB helicase stimulation by wild-type and various TFIIE? mutants.
XPB is in a different context when incorporated into the multi-subunit TFIIH, which contains nine other polypeptides, including its binding partner p52 and another helicase, XPD. We wished to learn whether TFIIE? would still stimulate XPB helicase activity in this context. To avoid a signal from the XPD helicase activity, an XPB-specific 3'–5' helicase substrate was used (21,22).
Figure 3A shows that the isolated beta subunits retain the ability to stimulate the XPB helicase in the context of intact TFIIH (compare lane 8 with lane 7 and compare lane 6 with lane 5), although the stimulation is less using this assay (2-fold). Figure 3B shows that TFIIE? also stimulates the ATPase activity of TFIIH (lane 4 versus lane 3), as would be expected based on the property of isolated XPB (see above). We conclude that TFIIE? stimulates the enzymatic activities of XPB, both in isolated form and in the context of TFIIH.
Figure 3 TFIIE? stimulates TFIIH helicase and ATPase activity. (A) Helicase assay. Lane 1, XPB 3'–5' helicase specific substrate; lane 2, substrate was heated for 1 min; lane 3, 5 and 7 contained 30 ng of TFIIH and the reaction times were 15, 30 and 45 min, respectively; lane 4, 6 and 8 contained 80 ng of TFIIE? and 30 ng of TFIIH and the reaction times are 15, 30 and 45 min, respectively. (B) ATPase assay. Lane 1, ATP alone; lane 2, 80 ng of TFIIE? was added; lane 3, 60 ng of TFIIH was added; lane 4 contained 80 ng of TFIIE? and 60 ng of TFIIH. The ATPase stimulation was 2.2 ± 0.4-fold.
TFIIE counteracts TFIIE? stimulation of the XPB helicase
In contrast to the above results, the TFIIE complex, consisting of an 2?2 tetramer, is known to have the capacity to negatively regulate the helicase activity of XPB and TFIIH (14,39). This negative regulation is lessened in the presence of an antibody to the alpha subunit (39). These observations raise the possibility that the beta subunit has stimulation potential but in the context of intact TFIIE it can be moderated by the inhibitory potential of the alpha subunit. To test directly whether alpha can inhibit the stimulatory effect of beta, TFIIE was allowed to form a complex with TFIIE? before the addition of XPB and its helicase substrate. As alpha is titrated against beta, the stimulatory activity of beta falls progressively (Figure 4A, lanes 3–5 and summarized in Figure 4B), confirming the moderation effect of the alpha subunit of the helicase stimulation by the beta subunit.
Figure 4 TFIIE prevents TFIIE? from stimulating XPB. (A) Helicase assay. Lane 1, substrate alone; lane 2, XPB was added; lane 3, 0.8 μg of TFIIE? and XPB were included. For lanes 4 and 5, 1.6 and 3.2 μg, respectively, of IIE were incubated with 0.8 μg of TFIIE? for 15 min before the addition of XPB and the substrate. (B) A graphical representation of the helicase activities of samples in lanes 3–5.
TFIIE inhibits the XPB helicase directly, rather than working through TFIIE?
We tested the possibility that TFIIE moderates the TFIIE? helicase stimulation through its interaction with TFIIE?. Two N-terminal deletion mutants of TFIIE were constructed (Figure 5A) that would be expected to prevent the formation of a complex with TFIIE? (16). The CoIP experiment in Figure 5B confirms that the wild-type TFIIE binds TFIIE? (lane 2) but that N-terminal truncations of amino acids 1–49 (lane 3) or 1–66 (lane 4) prevent the association of TFIIE with TFIIE?.
Figure 5 TFIIE binds to XPB to counteract TFIIE? stimulation. (A) Western blot of wild-type TFIIE and its mutants. (B) CoIP of TFIIE? with wild-type TFIIE and its mutants. (C) Helicase assay. Lane 3, XPB was added; lane 4, TFIIE? and XPB were added (70% of substrate was unwound); lanes 5 and 6 as in lane 4, except 2.4 μg of TFIIE or 50–439 were added, respectively (30% of substrate were unwound for both).
The non-interacting mutant 50–439 was then tested for the ability to inhibit TFIIE?-stimulated XPB helicase activity. Figure 5C shows that the inhibition is still intact, even though the mutant TFIIE can no longer bind TFIIE?. The inhibition by the non-interacting alpha mutant is comparable with that of wild-type TFIIE (compare lanes 5 and 6 with the stimulated control in lane 4). We conclude that the ability of TFIIE to inhibit stimulation of XPB helicase activity does not depend on its association with TFIIE?. Apparently, TFIIE? and TFIIE can act independently directly on XPB, one to stimulate and the other to inhibit.
TFIIE? mutants defective in the stimulation of the XPB helicase are also defective in transcription
To examine the physiological relevance of the IIE beta stimulation of the XPB helicase, we attempted to identify and test transcription of IIE beta mutants that would be specifically defective in helicase stimulation. The data of Figure 2 showed that the deletion of the C-terminal BR-HL region lessens helicase stimulation and further deletion through the LR sigma 3 region reduces the residual activity, implicating multiple regions. The C-terminal BR-HLH is dense with overlapping functions (38). These include the IIE–alpha interaction (36,38) that is presumed to be important for recruitment. Therefore, mutants in this region would be expected to alter multiple interactions, not just stimulation of the XPB helicase. In contrast, no functions have yet been ascribed to the LR sigma 3 region, although it is known that its deletion results in a loss of basal transcription activity on a linearized template (36).
Several mutants were made in this region. These changed highly conserved hydrophobic residues to the hydrophilic serine (I171S and I189S). The extract was immunodepleted so as to make transcription depend on added wild-type TFIIE (44); this was reproduced with wild-type TFIIE (Figure 6A, depleted lane 1 versus lane 2 with TFIIE added back). In contrast, transcription was strongly (Figure 6A, lane 3 versus lane 2) or partially (lane 4 versus lane 2) defective when the mutant forms of TFIIE were added back. These two mutants were also found to be associated with defects in stimulated XPB helicase activity (Figure 6B, lanes 3 and 4 versus the wild-type in lane 2). Defects in XPB helicase activity are known to be led to defects in transcription (5,28), so the transcription defect associated with these TFIIE mutants is most likely due to lower XPB helicase activity (Figure 6A and B), although other contributions cannot be excluded.
Figure 6 Mutants that fail to stimulate XPB helicase activity also have transcription defects. (A) In vitro transcription at the AdML promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIE? and TFIIE; lane 3, with mutant I171S and TFIIE (0% transcription activity compared with wild-type); lane 4, with mutant I189S and TFIIE (60% transcription activity compared with wild-type). (B) Helicase assay. Lane 1, 2 μg of XPB; lane 2, XPB and 1.2 μg of wild-type TFIIE? (3-fold stimulation); lane 3, XPB and 1.2 μg of I171S; lane 4, XPB and 1.2 μg of I189S. Only the released oligo is shown. (C) CoIP with TFIIE? and general transcription factors. Top panel: lane 1, anti-FLAG resin plus wild-type TFIIE?; lanes 2–4 anti-FLAG resin pre-bound with FLAG-tagged TFIIE plus wild-type TFIIE or its mutants. Middle panel: lane 1, glutathione resin with GST plus wild-type TFIIE?; lanes 2–4 glutathione resin with GST–XPB plus wild-type TFIIE? or its mutants. Bottom panel: lane 1, anti-FLAG resin plus wild-type TFIIE? with His6 tag; lanes 2–4, anti-FLAG resin pre-bound with FLAG-tagged wild type TFIIE? 6x His tag or its mutants. (D) Pre-initiation complex formation of wild-type TFIIE? and its mutants. Lane 1, beads with equimolar of the template without the E4 promoter and gal sites are incubated with TFIIE? depleted extract complement with recombinant wild-type TFIIE. Lanes 2–4, beads bound to G9E4 template are incubated with recombinant wild-type TFIIE, I171S or I189S, respectively. The left panel indicates the transcription factors that are being probed. (E) In vitro transcription at the G9E4 promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIE; lane 3, with mutant I171S and TFIIE; lane 4, with mutant I189S and TFIIE.
Several other in vitro functions were found to be normal using a CoIP assay (8). First, the two beta mutants bind TFIIE comparably with wild-type (Figure 6C, top row, compare mutants in lanes 3 and 4 with the wild-type in lane 2). Second, the beta subunits are known to dimerize as part of intact TFIIE (11) and this beta dimerization function is normal (Figure 6C, bottom panel). Third, the binding of beta to XPB is also comparable for the mutants and wild-type (Figure 6C, middle panel).
Although this region would not be expected to affect pre-initiation complex formation, as the C-terminus binds the alpha subunit presumably required for assembly, this was tested directly using an immobilized template pull-down assay with a biotinylated promoter (42). A G9E4 template was chosen over the AdML G-less cassette template used earlier because the G-less cassette appeared to bind a significant amount of factors non-specifically (data not shown). The wild-type transcription complex was shown to be functional as hydrolysable NTPs resulted in polymerase II and TFIIH leaving the complex (data not shown) (42).
Figure 6D shows that transcription factors were recruited to the template, whether the wild-type or the mutant forms of beta were present. These include the RNA polymerase itself (second to bottom row) and two subunits of TFIIH, XPB (top row) and p62 (bottom row); the control in this case was the use of promoter-less DNA (compare this control in column 1 with promoter DNA pull-downs in columns 2–4). Although the wild-type and mutant TFIIE subunits were also pulled down comparably, we could not confirm the promoter specificity of the interactions (again compare with column 1 in this context).
Overall, the data demonstrate that the TFIIE? mutants recruit TFIIH to the template but are defective in stimulating the TFIIH XPB helicase activity. The associated transcription defect is re-confirmed using the same E4 template used in the template pull-down assay (lanes 3 and 4 versus lane 2 of Figure 6E), which complements the transcription data for the ML template (Figure 6A).
DISCUSSION
The ATP-dependent XPB helicase activity of TFIIH is used in triggering promoter-bound RNA polymerase II to progress through initiation and into early elongation (27,28,30,31,34). The enzyme activity in vitro is known to be very low; contributions may include the lack of a binding partner, such as p44, which stimulates the TFIIH XPD helicase (25,26) and inhibition by the alpha subunit of TFIIE (39). In this paper, we show that the ATPase and helicase activities of XPB can be strengthened by its partnering with the beta subunit of TFIIE. This stimulation appears to be functionally significant as point mutations in TFIIE? that have defects in XPB stimulation are also defective in transcription; in contrast, the mutations do not interfere with the recruitment of XPB to the transcription complex or with TFIIE? binding to TFIIE.
The stimulatory activity of the TFIIE? subunit can be counter-acted by the inhibitory activity of its alpha partner subunit. The current data show that the stimulatory activity of TFIIE? and the inhibitory activity of TFIIE act independently, i.e. each works directly on XPB. This suggests that the TFIIE complex may be built so that it has the potential to either activate or inhibit. Photo-cross-linking data (46) suggest that ATP causes TFIIE to re-arrange so as to better engage the downstream region of the transcription bubble (from +4 to +8) during promoter opening, while no accompanying change in the positioning of the beta subunit was detected. It is possible that this re-arrangement within TFIIE releases the beta subunit to stimulate the ATP-dependent XPB helicase to enhance the subsequent promoter escape steps. The data show that it is the C-terminus of TFIIE? that accomplishes this. Because this region interacts with many components of the transcription complex (36,38), the data raise the possibility that it has a role in multiple re-modeling interactions subsequent to complex assembly (47–49).
Among RNA polymerases, only RNA polymerase II requires a helicase to transit to elongation mode following recruitment to the promoter (50–52), and TFIIH and TFIIE have evolved to meet this unique requirement. This mechanism enhances the potential diversity of regulation and factors may exist that target the TFIIE? helicase stimulatory activity to control the entry of the promoter-bound RNA polymerase into elongation phase. This transition involves the opening of DNA around the start site (7,52) and the release of factors holding RNA polymerase to the promoter (48,49), leading to the addition of factors that couple transcription elongation and RNA processing (47). These steps cannot occur without the action of the ATP-dependent XPB helicase (13,30,31,36,38), which the current data suggest that it is controlled by TFIIE?. Such considerations raise the possibility that any or all steps could be potential targets for regulation via the TFIIE?-stimulated ATP-dependent helicase activity, although the recent demonstration that helicase action comes into play directly after promoter opening (Y. C. Lin and J. D. Gralla, accepted by Nature Structural and Molecular Biology) favors a primary role in providing the energy needed to re-model open pre-initiation complexes and drive RNA polymerase into elongation mode.
ACKNOWLEDGEMENTS
The authors thank David Vargas for purifying some of the proteins and for technical assistance. The authors are grateful to Hao Duong (UCLA, Department of Chemistry and Biochemistry) for providing TFIIE and TFIIE? during the initial phase of these studies, Dr Jean-Marc Egly for the XPB clone and Dr Mike Carey for materials and protocols for immobilized template assay. This research was supported by USPHS grant NIHGM49048. Funding to pay the Open Access publication charges for this article was provided by USPHS.
REFERENCES
Hampsey, M. (1998) Molecular genetics of the RNA polymerase II general transcriptional machinery Microbiol. Mol. Biol. Rev., 62, 465–503 .
Orphanides, G., Lagrange, T., Reinberg, D. (1996) The general transcription factors of RNA polymerase II Genes Dev., 10, 2657–2683 .
Lee, T.I. and Young, R.A. (2000) Transcription of eukaryotic protein-coding genes Annu. Rev. Genet., 34, 77–137 .
Buratowski, S., Hahn, S., Guarente, L., Sharp, P.A. (1989) Five intermediate complexes in transcription initiation by RNA polymerase II Cell, 56, 549–561 .
Dvir, A., Conaway, J.W., Conaway, R.C. (2001) Mechanism of transcription initiation and promoter escape by RNA polymerase II Curr. Opin. Genet. Dev., 11, 209–214 .
Holstege, F.C., Fiedler, U., Timmers, H.T. (1997) Three transitions in the RNA polymerase II transcription complex during initiation EMBO J., 16, 7468–7480 .
Holstege, F.C., van der Vliet, P.C., Timmers, H.T. (1996) Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH EMBO J., 15, 1666–1677 .
Flores, O., Lu, H., Reinberg, D. (1992) Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH J. Biol. Chem., 267, 2786–2793 .
Wolner, B.S. and Gralla, J.D. (1997) Promoter activation via a cyclic AMP response element in vitro J. Biol. Chem., 272, 32301–32307 .
Spangler, L., Wang, X., Conaway, J.W., Conaway, R.C., Dvir, A. (2001) TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA Proc. Natl Acad. Sci. USA, 98, 5544–5549 .
Peterson, M.G., Inostroza, J., Maxon, M.E., Flores, O., Admon, A., Reinberg, D., Tjian, R. (1991) Structure and functional properties of human general transcription factor IIE Nature, 354, 369–373 .
Park, E., Guzder, S.N., Koken, M.H., Jaspers-Dekker, I., Weeda, G., Hoeijmakers, J.H., Prakash, S., Prakash, L. (1992) RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability Proc. Natl Acad. Sci. USA, 89, 11416–11420 .
Goodrich, J.A. and Tjian, R. (1994) Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II Cell, 77, 145–156 .
Inostroza, J., Flores, O., Reinberg, D. (1991) Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of general transcription factor IIE J. Biol. Chem., 266, 9304–9308 .
Ohkuma, Y., Sumimoto, H., Horikoshi, M., Roeder, R.G. (1990) Factors involved in specific transcription by mammalian RNA polymerase II: purification and characterization of general transcription factor TFIIE Proc. Natl Acad. Sci. USA, 87, 9163–9167 .
Ohkuma, Y., Hashimoto, S., Wang, C.K., Horikoshi, M., Roeder, R.G. (1995) Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure–function studies of TFIIE-alpha Mol. Cell. Biol., 15, 4856–4866 .
Keriel, A., Stary, A., Sarasin, A., Rochette-Egly, C., Egly, J.M. (2002) XPD mutations prevent TFIIH-dependent transactivation by nuclear receptors and phosphorylation of RARalpha Cell, 109, 125–135 .
Schultz, P., Fribourg, S., Poterszman, A., Mallouh, V., Moras, D., Egly, J.M. (2000) Molecular structure of human TFIIH Cell, 102, 599–607 .
Ohkuma, Y. and Roeder, R.G. (1994) Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation Nature, 368, 160–163 .
Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J.H., Chambon, P., Egly, J.M. (1993) DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor Science, 260, 58–63 .
Schaeffer, L., Moncollin, V., Roy, R., Staub, A., Mezzina, M., Sarasin, A., Weeda, G., Hoeijmakers, J.H., Egly, J.M. (1994) The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor EMBO J., 13, 2388–2392 .
Roy, R., Schaeffer, L., Humbert, S., Vermeulen, W., Weeda, G., Egly, J.M. (1994) The DNA-dependent ATPase activity associated with the class II basic transcription factor BTF2/TFIIH J. Biol. Chem., 269, 9826–9832 .
Svejstrup, J.Q., Vichi, P., Egly, J.M. (1996) The multiple roles of transcription/repair factor TFIIH Trends Biochem. Sci., 21, 346–350 .
Serizawa, H., Conaway, J.W., Conaway, R.C. (1994) An oligomeric form of the large subunit of transcription factor (TF) IIE activates phosphorylation of the RNA polymerase II carboxyl-terminal domain by TFIIH J. Biol. Chem., 269, 20750–20756 .
Coin, F., Marinoni, J.C., Rodolfo, C., Fribourg, S., Pedrini, A.M., Egly, J.M. (1998) Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH Nature Genet., 20, 184–188 .
Dubaele, S., Proietti De Santis, L., Bienstock, R.J., Keriel, A., Stefanini, M., Van Houten, B., Egly, J.M. (2003) Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients Mol. Cell, 11, 1635–1646 .
Hwang, J.R., Moncollin, V., Vermeulen, W., Seroz, T., van Vuuren, H., Hoeijmakers, J.H., Egly, J.M. (1996) A 3'5' XPB helicase defect in repair/transcription factor TFIIH of xeroderma pigmentosum group B affects both DNA repair and transcription J. Biol. Chem., 271, 15898–15904 .
Tirode, F., Busso, D., Coin, F., Egly, J.M. (1999) Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD and cdk7 Mol. Cell, 3, 87–95 .
Douziech, M., Coin, F., Chipoulet, J.M., Arai, Y., Ohkuma, Y., Egly, J.M., Coulombe, B. (2000) Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein–DNA photo-cross-linking Mol. Cell. Biol., 20, 8168–8177 .
Fukuda, A., Nogi, Y., Hisatake, K. (2002) The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation Proc. Natl Acad. Sci. USA, 99, 1206–1211 .
Moreland, R.J., Tirode, F., Yan, Q., Conaway, J.W., Egly, J.M., Conaway, R.C. (1999) A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II J. Biol. Chem., 274, 22127–22130 .
Jawhari, A., Laine, J.P., Dubaele, S., Lamour, V., Poterszman, A., Coin, F., Moras, D., Egly, J.M. (2002) p52 Mediates XPB function within the transcription/repair factor TFIIH J. Biol. Chem., 277, 31761–31767 .
Winkler, G.S., Sugasawa, K., Eker, A.P., de Laat, W.L., Hoeijmakers, J.H. (2001) Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF Biochemistry, 40, 160–165 .
Bradsher, J., Coin, F., Egly, J.M. (2000) Distinct roles for the helicases of TFIIH in transcript initiation and promoter escape J. Biol. Chem., 275, 2532–2538 .
Maxon, M.E., Goodrich, J.A., Tjian, R. (1994) Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance Genes Dev., 8, 515–524 .
Okamoto, T., Yamamoto, S., Watanabe, Y., Ohta, T., Hanaoka, F., Roeder, R.G., Ohkuma, Y. (1998) Analysis of the role of TFIIE in transcriptional regulation through structure–function studies of the TFIIE? subunit J. Biol. Chem., 273, 19866–19876 .
Chen, J., Larochelle, S., Li, X., Suter, B. (2003) Xpd/Ercc2 regulates CAK activity and mitotic progression Nature, 424, 228–232 .
Watanabe, T., Hayashi, K., Tanaka, A., Furumoto, T., Hanaoka, F., Ohkuma, Y. (2003) The carboxy terminus of the small subunit of TFIIE regulates the transition from transcription initiation to elongation by RNA polymerase II Mol. Cell. Biol., 23, 2914–2926 .
Drapkin, R., Reardon, J.T., Ansari, A., Huang, J.C., Zawel, L., Ahn, K., Sancar, A., Reinberg, D. (1994) Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II Nature, 368, 769–772 .
Yan, M. and Gralla, J.D. (1997) Multiple ATP-dependent steps in RNA polymerase II promoter melting and initiation EMBO J., 16, 7457–7467 .
van Vuuren, A.J., Vermeulen, W., Ma, L., Weeda, G., Appeldoorn, E., Jaspers, N.G., van der Eb, A.J., Bootsma, D., Hoeijmakers, J.H., Humbert, S., et al. (1994) Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH) EMBO J., 13, 1645–1653 .
Johnson, K.M., Wang, J., Smallwood, A., Carey, M. (2004) The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly Methods Enzymol., 380, 207–219 .
Dignam, J.D., Lebovitz, R.M., Roeder, R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei Nucleic Acids Res., 11, 1475–1489 .
Park, C.H., Mu, D., Reardon, J.T., Sancar, A. (1995) The general transcription-repair factor TFIIH is recruited to the excision repair complex by the XPA protein independent of the TFIIE transcription factor J. Biol. Chem., 270, 4896–4902 .
Evans, E., Moggs, J.G., Hwang, J.R., Egly, J.M., Wood, R.D. (1997) Mechanism of open complex and dual incision formation by human nucleotide excision repair factors EMBO J., 16, 6559–6573 .
Kim, T.K., Ebright, R.H., Reinberg, D. (2000) Mechanism of ATP-dependent promoter melting by transcription factor IIH Science, 288, 1418–1422 .
Svejstrup, J.Q. (2004) The RNA polymerase II transcription cycle: cycling through chromatin Biochim. Biophys. Acta, 1677, 64–73 .
Yudkovsky, N., Ranish, J.A., Hahn, S. (2000) A transcription reinitiation intermediate that is stabilized by activator Nature, 408, 225–229 .
Yean, D. and Gralla, J.D. (1999) Transcription reinitiation rate: a potential role for TATA box stabilization of the TFIID:TFIIA:DNA complex Nucleic Acids Res., 27, 831–838 .
Paule, M.R. and White, R.J. (2000) Survey and summary: transcription by RNA polymerases I and III Nucleic Acids Res., 28, 1283–1298 .
Geiduschek, E.P. and Kassavetis, G.A. (2001) The RNA polymerase III transcription apparatus J. Mol. Biol., 310, 1–26 .
Wang, W., Gralla, J.D., Carey, M. (1992) The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA Genes Dev., 6, 1716–1727 .(Yin C. Lin and Jay D. Gralla*)