From the Howard Hughes Medical Institute, Division of Nucleic Acid Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635
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ABSTRACT |
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A procedure to immunoaffinity purify the human transcription factor IIH (TFIIH) was developed using a monoclonal antibody that recognizes an epitope in ERCC3 (XPB), the largest subunit of TFIIH. The epitope recognized by the monoclonal antibody was mapped to 20 amino acids. A peptide containing the epitope was capable of displacing TFIIH from an immunoaffinity column containing the monoclonal antibody. The immunoaffinity purification procedure described allows a simple and efficient method to purify both the "core" and "holo" TFIIH complexes.
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INTRODUCTION |
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Initiation of transcription by RNA polymerase II (RNAPII) is a complex process requiring the assistance of five factors known as the general transcription factors (GTFs). These are transcription factor (TF)1 IIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). TFIID is a multisubunit complex that includes the TATA binding protein (TBP) (3). TBP, in its recombinant form or as part of the TFIID complex, recognizes the TATA motif present in many genes and can nucleate the formation of transcription-competent complexes (4). The TBP (TFIID)-TATA complex provides the foundation for the entry of TFIIB, which is then followed by the entry of RNA polymerase II. RNA polymerase II is escorted to the promoter by TFIIF (5). The resulting DNA-protein complex, TBPolF, contains RNA polymerase II stably associated with the promoter (2). This complex, however, is unique with respect to complexes formed with other RNA polymerases since the formation of an RNA polymerase II transcription-competent complex requires the function of two other GTFs, TFIIE and TFIIH (6).
TFIIE is a tetrameric factor composed of two subunits, p56 and p34 (7, 8). TFIIE interacts with RNA polymerase II (9) and TFIIH (10, 11). It is currently unknown whether TFIIE enters the transcription complex with RNA polymerase or in a subsequent step with TFIIH. However, it is clear that the association of TFIIE with the TBPolF complex is necessary for the entry of TFIIH to the transcription complex (6). Recent studies have suggested an alternative model for the formation of transcription competent complexes. These studies suggest that the GTFs and RNA polymerase II are preassembled in an RNA polymerase holoenzyme complex (12-14).
TFIIH also known as BTF2 was initially purified as a factor required to
reconstitute accurate transcription by RNA polymerase II in
vitro (6, 15). BTF2 was originally presumed to be TFIIE (16).
However, the availability of antibodies against a subunit of TFIIE (17)
revealed that BTF2 and TFIIE are distinct (18). Furthermore, antibodies
generated against the 62-kDa subunit of BTF2 revealed that BTF2 and
TFIIH are the same factor (18, 19). Additionally, an activity isolated
from rat liver extracts, referred to as factor , contained a subunit
composition and properties similar to human TFIIH (20). Purification of
TFIIH demonstrated that it is a multisubunit factor composed of nine
polypeptides (6, 15, 20). The studies with the rat liver-derived factor were the first to demonstrate that factor
(TFIIH) copurified with a
DNA-stimulated ATPase activity. The authors also reported that the
ATPase activity of TFIIH was preferentially stimulated by the TATA
motif (20).
The subunit composition of TFIIH has proved to be intriguing. The cDNA clones encoding the different subunits of TFIIH have been isolated. The largest subunit (p89) was identified as the DNA excision repair protein ERCC3 (Excision Repair Cross Complement) (21). Mutations in the ERCC3 gene are responsible for the DNA repair defects observed in patients with xeroderma pigmentosum (XP) group B and Cockayne's syndrome (reviewed in Refs. 22 and 23). In addition, ERCC3 was found to contain a DNA-dependent ATPase and a 3' to 5' helicase activity. Subsequent studies demonstrated that the second largest subunit of TFIIH (p80) was encoded by the ERCC2 gene (11, 24, 25), which corrects the DNA repair defect in cultured cells obtained from patients with XP group D (reviewed in Refs. 22 and 23). ERCC2 possesses a 5' to 3' ATP-dependent DNA helicase activity. Mutations in the yeast TFIIH subunits that are homologous to p62 (TFB1) and p44 (hSSL1) demonstrated that these subunits also perform essential functions in the nucleotide excision repair process (26, 27). Moreover, the 34-kDa subunit of TFIIH has homology with domains of hSSL1, suggesting that it also participates in nucleotide excision repair (28). In agreement with these observations was the finding that the TFIIH complex is essential for nucleotide excision repair (29-31). Most recently, the p52 subunit of TFIIH was cloned (32). This subunit was found to be a homolog of Tfb2 a subunit of the yeast TFIIH (33). This subunit is also thought to be involved in repair because Tfb2 mutants in yeast have a UV-sensitive phenotype, and antibodies raised against the human protien inhibit nucleotide excision repair in microinjection assays (32, 33).
TFIIH also copurifies with a kinase activity specific for the carboxyl-terminal domain (CTD) of RNAPII (19, 34, 35). The CTD is composed of multiple, tandemly repeated copies of the heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The kinase activity of mammalian TFIIH was shown to reside in the cdk-activating kinase (CAK) complex composed of the catalytic subunit cdk7 and its regulatory subunits cyclin H and MAT1 (36-39). The cdk7 kinase activity was previously shown to be necessary for the activation of at least three cyclin-dependent kinases that regulate cell-cycle progression through G1 (cdk2 and cdk4) and mitosis (cdc2) (40). These findings suggested that TFIIH not only participates in transcription and nucleotide excision repair, but may also regulate cell cycle progression.
Studies performed initially in yeast (41), and recently extended to the mammalian factor (42, 43), demonstrate that TFIIH exists in at least two subcomplexes, a complex containing the core subunits of TFIIH (ERCC3, ERCC2, p62, p52, p44, and p34) that is devoid of the kinase complex, referred to as core-TFIIH, and a complex associated with the kinase complex (cdk7, cyclin H and MAT-1), referred to as holo-TFIIH. Interestingly, studies performed in HeLa cells have demonstrated that TFIIH is limiting with respect to CAK and that no more than 20% of the total cellular CAK is associated with the TFIIH complex (42). The studies with the human CAK demonstrated that it exists in three distinct complexes: CAK, a novel ERCC2-CAK complex, and TFIIH (42, 43). Moreover, these studies also demonstrated that the ERCC2-CAK complex, and not CAK, was capable of complementing the severely compromised transcription activity of core-TFIIH in vitro (42, 43).
As indicated above TFIIH is a complex factor composed of nine subunits. Conventional purification procedures for mammalian TFIIH generally consist of eight-ten column schemes (15, 20, 42), which are time consuming and often result in poor recovery. In this report, we describe a quick and efficient method to immunopurify functional TFIIH complexes.
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MATERIALS AND METHODS |
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Production of Hybridomas and Monoclonal Antibodies (mAbs) against the Largest Subunit of TFIIH-- Hybridomas producing ERCC3 and GST antibodies were prepared by Bios-Chile I.G.S.A. Immunization was carried out with a bacterially produced MBP-ERCC3 fusion protein or recombinant GST. The recombinant MBP-ERCC3 polypeptide was isolated by maltose affinity purification as described previously (44). The GST protein was purified using glutathione affinity chromatography. Isotyping was performed with a mouse antibody isotyping kit, Isostrip (Boehringer Mannheim).
Production of Truncated ERCC3 Proteins-- The MBP-ERCC3 fusion protein containing amino acids 199 to 782 of ERCC3 was generated by subcloning ERCC3 cDNA (nucleotides 689 to 2751) into pMAL-c2 vector (New England Biolabs). The clones encoding C-terminal truncations of ERCC3, extending from amino acids 199 to 497, or 199 to 355, were constructed by digesting the DNA encoding MBP-ERCC3 with PstI or SphI, respectively. PstI cleaves at nucleotide 1586, whereas SphI cleaves at nucleotide 1160. A XbaI linker containing stop codons in all three reading frames was ligated at the cleavage sites. To generate the clones encoding C-terminal truncations of ERCC3 extending to amino acids 303 and 242, oligonucleotide primers for PCR amplification were designed. These primers contain 20 nucleotides complementary to sequences at the 5'-end (ending at nucleotide 689) and 3'-end (ending at residues 1003 and 820 for truncations at amino acids 303 and 242, respectively). The 5'-end primer was flanked with a noncomplementary BamHI restriction enzyme site followed by eight noncomplementary bases to assist restriction enzyme cleavage of the PCR product. The 3'-end primers were flanked by a HinDIII restriction enzyme site. PCR reactions were carried out as follows: 10 ng of template DNA, 50 pmol of 3'-end primer, 50 pmol of 5'-end primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1× PCR buffer (Perkin-Elmer) and 5 units of Taq polymerase (Boehringer Mannheim). The PCR reactions (100 µl) were performed for 30 cycles using a model 9600 thermocycler (Perkin-Elmer) PCR system. The PCR products were purified by phenol/chloroform extraction and cleaved with BamHI and HinDIII restriction endonucleases. The expression vector pMAL-c2 was also cleaved with BamHI and HinDIII in the polylinker region followed by alkaline phosphatase treatment. The various PCR products were ligated into the pMAL-c2 expression vector with T4 DNA ligase (New England Biolabs).
In the pMAL-c2 expression system, the cloned gene is inserted downstream of the MAL-E gene in the same reading frame. The MAL-E gene encodes the maltose binding protein (MBP) and allows one-step purification using a maltose affinity column. All DNA constructs encoding truncated MBP-ERCC3 proteins were transformed into Escherichia coli DH5Immunoprecipitation Competition Experiments-- Four overlapping peptides extending from amino acids 242 to 303 of ERCC3 were synthesized. Peptide 1 extends from amino acids 242 to 261, peptide 2 from amino acids 256 to 275, peptide 3 from amino acids 270 to 291, and peptide 4 from amino acids 281 to 303. These peptides were used as competitors in immunoprecipitation experiments used to map the epitope of ERCC3 that is recognized by one of the monoclonal antibodies (mAb 3G4).
Monoclonal antibody 3G4 was immobilized on protein A-agarose as follows. A 1:1 (v/v) ratio of ascites containing anti-ERCC3 mAb to protein A-agarose beads (Boheringer Mannheim) in PBS, was incubated for 90 min at 25 °C with rotation. After extensive washing of the beads with PBS, the samples were divided into five separate tubes each containing 20 µl of the antibody-coupled beads. Each aliquot was then subjected to a 90-min incubation at 4 °C with a particular peptide (as indicated) or PBS. Next, 50 µl of a 0.5 M KCl-eluted phosphocellulose fraction (4 mg/ml) that is enriched in TFIIH (6) was added to each tube and incubated for an additional 90 min at 4 °C with rotation. The beads were then washed three times with IP wash buffer (20 mM HEPES, pH 7.5, 0.4 M NaCl, 1 mM PMSF, 0.5% Nonidet P-40). After a final wash with PBS to remove the Nonidet P-40, the immunoprecipitates were eluted by boiling in SDS gel loading buffer for 5 min. The eluate was run on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and analyzed by Western blot using antibodies recognizing subunits of TFIIH. The polypeptides were visualized using alkaline phosphatase-coupled secondary antibodies (Promega).Affinity Purification of Recombinant ERCC3 and Human TFIIH Complexes-- Anti-ERCC3 or anti-GST monoclonal antibodies were covalently coupled to protein A-agarose as described (45). The peptide used for the elutions of MBP-ERCC3 or TFIIH from the affinity columns consists of 24 amino acids containing residues 242 to 261 of ERCC3 flanked by two additional lysine residues at each end to increase its solubility.
To affinity purify MBP-ERCC3 with the mAb 3G4, an extract was prepared from E. coli DH5In Vitro Transcription and Kinase Assays-- Transcription assays contained the adenovirus major late promoter (200 ng) directing transcription of a G-less cassette as a template, recombinant human TBP (5 ng), TFIIB (5 ng), TFIIE (15 ng), and TFIIF (25 ng), and highly purified RNA polymerase II (500 ng) isolated from HeLa cells as described (13). Conventionally purified TFIIH used as a control in kinase assays was prepared as described in Ref. 38 except a Pharmacia Superose-6 HR 10/30 column was substituted for the Pharmacia Superdex-200 16/60 column. The transcriptionally active fractions from the Superose-6 column were pooled and used for these experiments. These fractions are devoid of all the other known GTFs or CTD kinases. Transcription assays were supplemented with conventionally purified XPD-CAK (5, 10, and 15 ng) derived from the ceramic hydroxyapatite column purification step as described in (42). Transcription assays were performed as described in Ref. 46. To obtain a complete dependence on TFIIH, the DNA was linearized. Kinase assays (30 µl) were performed as described (38) using a CTD peptide (4 µg) containing four copies of the heptapeptide repeat as a substrate.
Immunoblot Analysis-- All immunoblots were performed with nitrocellulose. Blots were incubated with primary antibodies, either mouse monoclonal or rabbit polyclonal, in TBS (20 mM Tris-HCl, pH 7.5, 0.2 mM NaCl) with 0.5% Tween-20 for 3 h at 25 °C. After extensive washing with TBS, 0.5% Tween-20, the blots were incubated with a 1:5000 dilution of either anti-mouse or anti-rabbit secondary antibody conjugated to alkaline phosphatase (Promega) for 2 h. Blots were extensively washed with TBS, 0.5% Tween-20 prior to development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
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RESULTS |
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Epitope Mapping of a Monoclonal Antibody Recognizing the Largest Subunit of TFIIH-- Monoclonal antibodies were generated against a bacterial expressed MBP-ERCC3 fusion protein that was purified by maltose affinity chromatography. Monoclonal antibodies were initially screened for their ability to recognize ERCC3 in Western blot analysis, and for their ability to immunoprecipitate ERCC3 along with other TFIIH subunits from HeLa cell nuclear extract. One ERCC3 mAb (3G4/E5) was selected which fulfilled both of these criteria (data not shown, see below).
We next screened this mAb for its ability to recognize a series of C-terminal truncated MBP-ERCC3 fusion proteins (Fig. 1a) by Western blot analysis (Fig. 1b). This analysis allowed us to identify the region in ERCC3 recognized by the monoclonal antibody. C-terminal truncated proteins extending up to amino acid 303 of ERCC3 were recognized by the mAb. However, a protein containing a C-terminal deletion ending at residue 242 was not recognized by the antibody (Fig. 1b). All polypeptides were recognized by polyclonal antibodies (Fig. 1b). Therefore, we concluded that the epitope recognized by the mAb 3G4 lies within a 51-amino acid segment located between amino acids 242 to 303 (Fig. 1a).
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Affinity Purification of Holo and Core-TFIIH Complexes-- Having established that the monoclonal antibody 3G4 could recognize the ERCC3 epitope within the multisubunit TFIIH complex (Fig. 2a) and that a peptide containing amino acids 242 to 261 of ERCC3 was capable of eluting recombinant ERCC3 from a column containing the monoclonal antibody, we attempted to affinity purify the human TFIIH complex.
A crude protein fraction derived from the second step of purification of human TFIIH was applied to both an anti-ERCC3 (3G4) and anti-GST control immunoaffinity column (see "Materials and Methods"). After allowing adequate time for binding, the columns were washed with a buffer containing 0.4 M KCl followed by a buffer containing 0.1 M KCl. Polypeptides bound to the column were eluted by incubation with peptide 1 for 1 h, and then proteins were recovered (for details, see "Materials and Methods"). The eluate was dialyzed to remove the peptide from the fraction prior to analysis. Silver staining of the eluate from the anti-ERCC3 column revealed the presence of at least eight polypeptides migrating with molecular weights consistent with that of the subunits of TFIIH (Fig. 3a). These polypeptides were absent in the eluate from the control column (Fig. 3a, compare lanes 3 and 4 with lanes 6 and 7). Western blot analysis confirmed that the eluate from the anti-ERCC3 column, but not that of the control column, contains the core-TFIIH subunits ERCC3, ERCC2, p62, and p44 as well as the CAK components cdk7, cyclin H, and MAT1 (Fig. 3b, and data not shown). The affinity purified TFIIH was transcriptionally active (see below) and free of other GTFs (data not shown).
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Functional Analysis of the Immunoaffinity Purified TFIIH Complexes-- We next analyzed the two immunoaffinity purified TFIIH complexes for their ability to direct transcription and to phosphorylate the CTD of RNA polymerase II.
A TFIIH-dependent transcription system was reconstituted using the AdMLP and recombinant TBP, TFIIB, TFIIE, TFIIF, and highly purified human RNA polymerase II (Fig. 4a). Consistent with previous observations, in the absence of TFIIH, no transcription was detected (lane 2). However, the affinity purified holo-TFIIH complex was capable of supporting transcription in the reconstituted system (lane 6). The ability of the affinity purified core-TFIIH complex was severely compromised (lane 10). Nonetheless, the addition of an XPD-CAK complex markedly stimulated transcription of the core-TFIIH complex (lanes 11-13). The XPD-CAK complex was unable to support transcription in the absence of core-TFIIH (lanes 3-5) and was without a significant effect when added to the holo-TFIIH complex (lanes 7-9). Equal molar quantities of the core-TFIIH and holo-TFIIH complexes were used in these assays based on p62 and ERCC3 immunoreactivities. We should also note that we attempted to restore the compromised transcription activity of the core-TFIIH with a purified CAK fraction that was devoid of ERCC2 and were unable to see any significant effect (data not shown). This result is in accordance with previous results (42, 43).
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DISCUSSION |
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The purpose of our studies was to develop a fast and efficient method to purify TFIIH. Due to the availability of the recombinant GTFs TBP, TFIIA, TFIIB, TFIIE, and TFIIF and the advent of affinity purification strategies to isolate RNA polymerase II and TFIID complexes (50-52), it has become relatively easy to set up highly defined mammalian transcription systems.
The multisubunit TFIIH complex (53, 54) may prove to be too complex to be reconstituted with recombinant proteins and is therefore currently purified from cell extracts. Conventional purification procedures for mammalian TFIIH generally consists of 8-10 column schemes (6, 15, 20), which is time consuming and plagued with poor recovery. The procedure presented in this paper provides a quick and efficient method to purify both the TFIIH holoenzyme and a core-TFIIH complex. This type of purification is essential to facilitate reconstitution of a defined transcription system. In this study, we used an extract prepared from HeLa cells to purify TFIIH; however, this method should also work to purify TFIIH from murine or bovine extracts since the epitope recognized by the ERCC3 mAb (3G4/E5) is conserved in these species. Unfortunately, this epitope is not conserved in Drosophila melanogaster or Saccharomyces cerevisiae.
Our observation that the CAK components were removable from the TFIIH complex by increasing the stringency of the wash buffer is in agreement with previous observations (38, 42, 43, 47). Addition of a purified fraction containing XPD-CAK to core-TFIIH was capable of restoring transcription activity. This result is in accordance with results recently published (42, 55). We also attempted to restore the transcription activity of our affinity purified core-TFIIH with a purified CAK fraction that was devoid of XPD and were unsuccessful (data not shown). The exact composition of the XPD-CAK complex has yet to be defined. Future studies with recombinant CAK complexes will be needed to define all of the components present in the XPD-CAK complex. It has been reported that the kinase activity of the CAK complex may not be required for transcription from all class II promoters (56, 57). These studies used the transient expression of an epitope-tagged mutant cdk7 protein to purify a TFIIH complex that was deficient in kinase activity. Although the complexes were devoid of the kinase activity, they contained the CAK components. The ability to reconstitute the XPD-CAK complex with recombinant and mutant proteins will decipher whether the requirement for XPD-CAK is only a structural one or whether different promoters have different requirements.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Sancar for providing the MBP-ERCC3 clone used for immunization and to generate truncations. We also thank Dr. G. Orphanides for critical reading of the manuscript. We acknowledge A. Jamett and P. Valenzuela from Bios-Chile I.G.S.A. and its subsidiary, Austral Biologicals, San Ramon, California, for producing the monoclonal antibodies and providing the ascitic fluids.
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FOOTNOTES |
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* This work was supported in part by a grant from the National Institute of Health (GM-37120) and the Howard Hughes Medical Institute (to D. R.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Predoctoral Training
Grant GM08360.
§ Partially supported by a grant from the New Jersey Commission on Cancer Research (90-2138-CCR-00).
¶ To whom correspondence should be addressed. Tel.: 908-235-4195; Fax: 908-235-5294; E-mail: reinbedf{at}umdnj.edu.
1 The abbreviations used are: TF, transcription factor; TBP, TATA binding protein; GTF, general transcription factor; XP, xeroderma pigmentosum; CTD, carboxyl-terminal domain; CAK, cdk-activating kinase; mAb, monoclonal antibody; GST, glutathione S-transferase; PCR, polymerase chain reaction; MBP, maltose binding protein; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.
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REFERENCES |
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