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J Biol Chem, Vol. 274, Issue 32, 22127-22130, August 6, 1999

COMMUNICATION
A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II^*

Rodney J. Moreland, Franck Tirode^§, Qin Yan^¶, Joan Weliky Conaway^¶**, Jean-Marc Egly^§, and Ronald C. Conaway

From the  Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the ^§ Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, B. P. 163, Illkirch, C.U. de Strasbourg, France, the ^¶ Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, and the  Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3'  5' DNA helicase catalyzed by its XPB subunit, a 5'  3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

TFIIH is a nine-subunit complex that possesses multiple catalytic activities, including DNA-dependent ATPase, DNA helicase, and a protein kinase that is capable of phosphorylating the carboxyl-terminal domain (CTD)¹ of the largest subunit of RNA polymerase II (1). The two largest TFIIH subunits are ATP-dependent DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes. The TFIIH-associated CTD kinase is a three-subunit subassembly, CDK-activating kinase (CAK), which is composed of the kinase/cyclin pair CDK7/cyclin H and the RING-H2 finger protein MAT1. TFIIH subunits are found in a variety of additional subassemblies, including a six-subunit complex (IIH6) containing XPB, XPD, p62, p52, p44, and p34, a five-subunit "core" complex (IIH5) containing XPB, p62, p52, p44, and p34, and a four-subunit XPD/CAK complex (2-6).

TFIIH was initially identified by its requirement in transcription initiation by RNA polymerase II (7). Initiation is an ATP-dependent process that requires at minimum the five general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (8, 9). Biochemical studies have shown that initiation in this minimal transcription system proceeds through multiple stages beginning with assembly of polymerase and all five general initiation factors into a closed preinitiation complex at the promoter (8, 9) and culminating in ATP-dependent formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts (10-13). Evidence supporting a role for TFIIH DNA helicase activity in ATP-dependent formation of the open complex was initially suggested by studies indicating that both TFIIH and ATP are dispensible for initiation by RNA polymerase II from artificial promoters containing premelted transcriptional start sites and from promoters on negatively supercoiled DNA templates (14-19).

In addition to its requirement in transcription initiation, TFIIH is also required for efficient promoter escape by RNA polymerase II (18, 20-22). Mechanistic studies have shown that a fraction of early RNA polymerase II elongation intermediates are prone to arrest at promoter-proximal sites in the absence of TFIIH or an ATP cofactor (18, 21-23). Circumstantial evidence that TFIIH DNA helicase activity is responsible for suppressing arrest of early elongation intermediates has come from the observation that promoter escape is blocked by the TFIIH DNA helicase inhibitor ATPS, but not by the TFIIH CTD kinase inhibitor H-8 (18).

Although evidence from previous studies suggested that TFIIH DNA helicase activity is required for ATP-dependent formation of the open complex and ATP-dependent promoter escape, a direct test of this hypothesis was not possible until sufficient quantities of purified TFIIH mutants lacking functional XPB or XPD DNA helicase were available. Recently, some of us (F. Tirode and J.-M. Egly) reported the development of methods for reconstitution of TFIIH and TFIIH subassemblies from wild type and mutant subunits (2, 4). By investigating the activities of TFIIH mutants, we observed that maximal TFIIH transcriptional activity requires all nine subunits, although the TFIIH subassembly IIH6 lacking CAK is active in ATP-dependent formation of the open complex and supports a reduced level of runoff transcription (4). In addition, by comparing the activities of IIH6 and two IIH6 mutants, IIH6/XPB-K346R and IIH6/XPD-K48R, which contain point mutations in the XPB and XPD ATP binding sites and lack DNA helicase activity (24, 25), we obtained evidence supporting the model that the XPB DNA helicase is essential for formation of the open complex and runoff transcription and that the XPD DNA helicase, though not essential, stimulates these reactions (2).

In this report, we exploit recombinant TFIIH mutants lacking functional XPB DNA helicase, XPD DNA helicase, or CAK to investigate the contribution of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape. Our findings argue that the XPB DNA helicase is primarily responsible for TFIIH action in suppression of arrest of early RNA polymerase II elongation complexes during their escape from the promoter.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Unlabeled ultrapure ribonucleoside 5'-triphosphates, 3'-O-MeGTP, and [-³²P]CTP (>3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Dinucleotides CpA and CpU, polyvinyl alcohol (type II) and -amanitin were obtained from Sigma. Acetylated bovine serum albumin and recombinant human placental ribonuclease inhibitor were from Promega.

Preparation of RNA Polymerase II and Transcription Factors-- RNA polymerase II and TFIIH were purified from rat liver nuclear extracts as described (26). Recombinant yeast TBP (27, 28), recombinant TFIIB (29), and recombinant TFIIF (30) were expressed in Escherichia coli and purified as described previously (27-30). Recombinant TFIIE was prepared as described previously (31), except that the 56-kDa subunit was expressed in E. coli strain BL21(DE3)-pLysS. IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R were expressed in Sf9 cells and purified through the heparin Ultrogel chromatography step as described previously (2). IIH6 and IIH6 mutants were further purified by anti-p44 immunoaffinity chromatography using the monoclonal antibody 1H5 (32). Recombinant CAK was purified as described previously (4).

Assay of Transcription-- Preinitiation complexes were assembled at the AdML promoter on the EcoRI to NdeI fragment of pDN-AdML (33) or on the premelted template fragment Ad(9/+1) (18) at 28 °C by a 45-60-min preincubation of 30-µl reaction mixtures containing 20 mM Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 4 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl alcohol, 3% (v/v) glycerol, 6 units of recombinant placental ribonuclease inhibitor, ~10 ng of DNA template fragment, ~5 ng of recombinant TBP, ~10 ng of recombinant TFIIB, ~10 ng of recombinant TFIIF, ~20 ng of recombinant TFIIE, 0.01 unit of RNA polymerase II, and, where indicated, ~100 ng of CAK and either equivalent amounts (~150 ng) of wild type IIH6 or IIH6 mutants or ~10 ng of rat TFIIH. Transcription was initiated by addition of 4 µl of a solution containing the nucleotides indicated in the figure legends. Reactions were stopped by addition of an equal volume of 9.0 M urea containing 0.025% (w/v) bromphenol blue and 0.025% (w/v) xylene cyanol FF. Transcripts were analyzed by electrophoresis through polyacrylamide gels containing 25% acrylamide, 3% bisacrylamide, 5.0 M urea, 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA. Transcription was quantitated using a Molecular Dynamics PhosphorImager.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To investigate the roles of the XPB and XPD DNA helicases and CAK in TFIIH-dependent promoter escape, we compared the abilities of IIH6 and two IIH6 mutants, IIH6/XPB-K346R and IIH6/XPD-K48R, which contain point mutations in the XPB and XPD ATP binding sites and lack DNA helicase activity (24, 25), to suppress arrest of early RNA polymerase II elongation intermediates in a minimal transcription system reconstituted with purified polymerase and general initiation factors TBP, TFIIB, TFIIE, and TFIIF. IIH6 and IIH6 mutants were expressed in Sf9 cells coinfected with baculoviruses encoding human TFIIH subunits p34, p44, p52, p62, wild type or mutant XPD, and wild type or mutant XPB (2). Recombinant IIH6 and IIH6 mutants were purified from lysates of Sf9 cells by sequential heparin ultrogel and anti-p44 immunoaffinity chromatography (2, 32). Recombinant CAK was purified from lysates of Sf9 cells coinfected with baculoviruses encoding CDK7, cyclin H, and MAT1 (4). The subunit compositions of wild type and mutant IIH6 complexes and CAK were verified by Western blotting, and the relative concentrations of wild type and mutant IIH6 complexes were estimated by quantitative Western blotting (Fig. 1 and data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Recombinant IIH6, IIH6 mutants, and CAK. A, structure of XPB and XPD. I-VI indicate the XPB and XPD helicase motifs. K346R and K48R indicate the positions of point mutations in XPB and XPD ATP binding sites, respectively. B, purified recombinant wild type (lane 2) or mutant IIH6 (lanes 3 and 4) or TFIIH purified from HeLa cells (heparin 5-PW fraction (41)) (lane 1) were separated by 12% SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies raised against each of the subunits. C, purified recombinant CAK was separated by 12% SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies against CDK7, cyclin H (CycH), and MAT1.

To characterize the transcriptional activities of IIH6 and IIH6 mutants, we began by using a dinucleotide-primed abortive initiation assay to compare their abilities to support transcription initiation from the AdML promoter in the minimal transcription system. In the presence of an ATP cofactor, RNA polymerase II will utilize dinucleotides to prime synthesis of promoter-specific transcripts (34). If only a dinucleotide primer and the next nucleotide encoded by the template are provided as substrates for transcription, polymerase will efficiently synthesize abortively initiated, trinucleotide transcripts (10, 35, 36). We and others have shown previously that abortive initiation from the AdML promoter can be measured in the presence of [-³²P]CTP and either initiating dinucleotide CpA or CpU, which prime transcription at positions 1 and 3 relative to the normal AdML transcriptional start site (Fig. 2A) (35, 37, 38). In addition, we and others have shown that maximal rates of abortive initiation from the AdML promoter depend strongly on an ATP cofactor and all five general initiation factors (21, 22, 38).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R in abortive initiation. RNA polymerase II preinitiation complexes were assembled at the AdML promoter as described under "Experimental Procedures." Equivalent amounts of wild type IIH6 or IIH6 mutants (normalized to XPB polypeptide) and ~100 ng of CAK were added to reactions, as indicated in the figure, 30 min prior to addition of nucleotides. A, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for the times indicated in the figure in the presence of 170 µM CpA, 5 µM ATP, and 15 µCi of [-32P]CTP. B, synthesis of abortive trinucleotide transcripts was carried out at 28 °C for 45 min in the presence of 170 µM CpA, 5 µM ATP, and 15 µCi of [-32P]CTP. Synthesis of trinucleotide transcripts was quantitated by PhosphorImager analysis.

As shown in Fig. 2A, wild type IIH6 stimulated the rate of abortive initiation above the low background level observed in the absence of TFIIH, whereas equivalent concentrations of the XPB mutant IIH6/XPB-K346R and the XPD mutant IIH6/XPD-K48R did not. CAK, which is composed of CDK7, cyclin H, and MAT1 subunits, detectably stimulated the rate of abortive initiation by both wild type IIH6 and the XPD mutant IIH6/XPD-K48R, but not by the XPB mutant IIH6/XPB-K346R (Fig. 2B). These findings are consistent with the results of Tirode et al. (2), who observed that the XPB mutant IIH6/XPB-K346R did not support detectable open complex formation and runoff transcription in the presence or absence of CAK, whereas the XPD mutant IIH6/XPD-K48R was substantially less active than IIH6, but could support a low level of runoff transcription that was stimulated by CAK.

To investigate the activities of IIH6 and IIH6 mutants in promoter escape, we took advantage of the artificial AdML promoter derivative Ad(9/1), which contains a premelted region from positions 9 to 1 relative to the normal transcriptional start site. The Ad(9/1) promoter supports transcription initiation by RNA polymerase II in the absence of TFIIH and an ATP cofactor and is therefore a useful model for investigating post-initiation roles of TFIIH and ATP (12, 16-18, 39). We previously observed that maximal synthesis of 18 nucleotide RNAs terminated at the first G residue of the Ad(9/1) transcript by incorporation of 3'-O-MeG requires TFIIH and ATP and is inhibited by ATPS (18). Further elongation of the 18-nucleotide transcript is independent of ATP and TFIIH; thus, RNA polymerase II elongation complexes that have completed synthesis of these transcripts can be considered to have escaped the promoter.

To compare the abilities of IIH6 and IIH6 mutants to support efficient promoter escape, RNA polymerase II preinitiation complexes were assembled at the Ad(9/1) promoter in the minimal transcription system in the presence of either IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Transcription was carried out in the presence of ATP or ATPS and the initiating dinucleotide CpU, UTP, [-³²P]CTP, and 3'-O-MeGTP. Reaction mixtures were then gel-filtered to remove unincorporated [-³²P]CTP and the large number of abortive transcripts synthesized during transcription of premelted templates (40), see also Fig. 3C).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Activities of IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R in promoter escape. RNA polymerase II preinitiation complexes were assembled at the premelted Ad(9/1) promoter as described under "Experimental Procedures." Equivalent amounts of wild type IIH6 and IIH6 mutants (normalized to XPB polypeptide) and ~100 ng of CAK were added to reactions, as indicated in the figure, 30 min prior to addition of nucleotides. A, initial transcribed region of Ad(9/+1) premelted promoter. B, synthesis of 18-nucleotide transcripts was carried out at 28 °C for 45 min in the presence of 170 µM CpU, 50 µM UTP, 50 µM 3'-O-MeGTP, 15 µCi of [-32P]CTP, and either 50 µM ATP or 50 µM ATPS. Ternary elongation complexes were purified by centrifugation through 300 µl of Sepharose CL-6B spin columns that had been equilibrated in transcription buffer. Synthesis of 18-nucleotide transcripts was quantitated by PhosphorImager analysis. C, synthesis of 18-nucleotide transcripts was carried out at 28 °C for 45 min in the presence of 170 µM CpU, 50 µM UTP, 50 µM 3'-O-MeGTP, 15 µCi of [-32P]CTP, and either 50 µM ATP or 50 µM ATPS.

As shown in Fig. 3B, in the presence of ATPS, the majority of RNA polymerase II elongation intermediates suffered arrest before completing synthesis of the 18 nucleotide, 3'-O-MeG-terminated transcript; similar levels of the 18-nucleotide transcript were synthesized whether reactions contained IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R. Substitution of ATP for ATPS increased accumulation of the 18-nucleotide transcript ~7-fold in reactions containing IIH6 and ~5-fold in reactions containing the XPD mutant IIH6/XPD-K48R. In contrast, substitution of ATP for ATPS had no significant effect on accumulation of the 18 nucleotide transcript in reactions containing the XPB mutant IIH6/XPB-K346R, arguing that the XPB DNA helicase makes a significantly greater contribution than the XPD DNA helicase to TFIIH function in ATP-dependent promoter escape.

As shown previously (2) and in Fig. 2B, the presence of the CAK subunits increases TFIIH activity in abortive initiation and in synthesis of runoff transcripts. To investigate the contribution of CAK to TFIIH-dependent promoter escape, IIH6 and IIH6 mutants were assayed in the presence and absence of CAK, and reaction products were analyzed without prior gel filtration. As shown in Fig. 3C, CAK had no detectable effect on the levels of 18 nucleotide transcripts synthesized in the presence of either wild type IIH6, IIH6/XPB-K346R, or IIH6/XPD-K48R, arguing that CAK does not contribute significantly to TFIIH-dependent promoter escape. Because these reactions were not gel-filtered, a large number of abortive transcripts can be observed. Nonetheless, the relative amounts of 18 nucleotide transcript synthesized in the presence of wild type IIH6, IIH6/XPB-K346R, and IIH6/XPD-K48R are comparable with those seen when reaction products were gel filtered prior to polyacrylamide gel electrophoresis (Fig. 3B, lanes 1, 3, and 5).

In summary, in this report we have taken advantage of recombinant TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. By comparing the activities of the TFIIH subassembly IIH6 and two IIH6 mutants, IIH6/XPB-K346R and IIH6/XPD-K48R, which contain point mutations in the XPB and XPD ATP binding sites and lack DNA helicase activity (24, 25), we have obtained evidence supporting the model that the XPB DNA helicase is primarily responsible for TFIIH action in ATP-dependent promoter escape. We observe (i) that the IIH6 point mutant IIH6/XPB-K346R, which contains wild type XPD DNA helicase but lacks functional XPB DNA helicase, is inactive in promoter escape and (ii) that the IIH6 point mutant IIH6/XPD-K48R, which contains wild type XPB DNA helicase but lacks functional XPD DNA helicase, supports promoter escape but less actively than wild type IIH6. Together with the recent findings of Tirode et al. (2), who presented evidence supporting the model (i) that the XPB DNA helicase is essential for ATP-dependent formation of the open complex and (ii) that the XPD DNA helicase stimulates this reaction, our results indicate that the relative contributions of the XPB and XPD DNA helicases to promoter escape closely parallel their contributions to open complex formation and suggest that TFIIH performs similar roles during both open complex formation and promoter escape.

	ACKNOWLEDGEMENT

We thank K. Jackson of the Molecular Biology Resource Center at the Oklahoma Center for Molecular Medicine for oligonucleotide synthesis.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant GM41628 (to R. C. C.), a Human Frontier Grant (to J. M. E.), and by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust.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.

^** Associate Investigator of the Howard Hughes Medical Institute.

To whom correspondence should be addressed. Tel.: 405-271-1950; Fax: 405-271-1580.

	ABBREVIATIONS

The abbreviations used are: CTD, carboxyl-terminal domain; 3'-O-MeGTP, 3'-O-methylguanosine 5'-triphosphate; ATPS, adenosine 5'-O-(thio)triphosphate; AdML, adenovirus 2 major late; CAK, CDK-activating kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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