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• W2019946452 abstract "Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH. Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH. Cyclin-dependent kinase 7 (CDK7) 1The abbreviation used is: CDK7, cyclin-dependent kinase 7; CAK, cdk-activating kinase; TF, transcription repair factor; RNAPII, RNA polymerase II; holo, holoenzyme; CTD, carboxyl-terminal domain; HTLV, human T-cell leukemia virus; TBP, TATA-binding protein; TB, TBP·TFIIB. was originally isolated as the catalytic subunit of the trimeric cdk-activating kinase (CAK) complex. This complex, consisting of CDK7, cyclin H, and MAT1, is responsible for activation of the mitotic promoting factor in vitro (1Fesquet D. Labbe J.-C. Derancourt J. Capony J.-P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.-C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar, 2Poon R.Y.C. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (333) Google Scholar, 3Solomon M.J. Harper J.W. Shuttleworth J. EMBO J. 1993; 12: 3133-3142Crossref PubMed Scopus (273) Google Scholar). The discovery that CDK7 was also a component of the basal transcription repair factor IIH (TFIIH) implicated a dual role for CDK7 in transcription as part of TFIIH and in the control of the cell cycle as the trimeric CAK complex (4Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 5Roy R. Adamczewski J.P. Seroz T. Vermuelen W. Tassan J.-P. Schaeffer L. Nigg E.A. Hoeijmakers J.H.J. Egly J.-M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 6Serizawa H. MäKelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar, 7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar). TFIIH is a multisubunit protein complex identified as a factor required for RNA polymerase II (RNAPII)-catalyzed transcription (8Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7356-7360Crossref PubMed Scopus (92) Google Scholar, 9Feaver W.J. Gileadi O. Kornberg R.D. J. Biol. Chem. 1991; 266: 19000-19005Abstract Full Text PDF PubMed Google Scholar, 10Gerard M. Fischer L. Moncollin V. Chipoulet J.-M. Chambon P. Egly J.-M. J. Biol. Chem. 1991; 266: 20940-20945Abstract Full Text PDF PubMed Google Scholar, 11Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Abstract Full Text PDF PubMed Google Scholar), and subsequently this complex was found to play a key role in nucleotide excision repair (12Feaver W.J. Svejstrup J.Q. Bardwell L. Bardwell A.J. Buratowski S. Gulyas K.D. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1993; 75: 1379-1387Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 13Schaeffer L. Roy R. Humbert S. Moncollin V. Vermuelen W. Hoeijmakers J.H.J. Chambon P. Egly J.-M. Science. 1993; 260: 58-63Crossref PubMed Scopus (666) Google Scholar, 14Drapkin R. Reardon J.T. Ansari A. Huang J.-C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Crossref PubMed Scopus (410) Google Scholar). At least nine polypeptides with molecular masses of 89, 80, 62, 52, 44, 40, 37, 36, and 34 kDa co-purify with mammalian TFIIH. The cDNAs encoding all of these subunits have now been cloned. p89 and p80 are the gene products of ERCC3 (XPB) and ERCC2 (XPD), respectively (13Schaeffer L. Roy R. Humbert S. Moncollin V. Vermuelen W. Hoeijmakers J.H.J. Chambon P. Egly J.-M. Science. 1993; 260: 58-63Crossref PubMed Scopus (666) Google Scholar, 14Drapkin R. Reardon J.T. Ansari A. Huang J.-C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Crossref PubMed Scopus (410) Google Scholar, 15Schaeffer L. Moncollin V. Roy R. Staub A. Mezzina M. Sarasin A. Weeda G. Vermuelen W. Hoeijmakers J.H.J. Egly J.-M. EMBO J. 1994; 13: 2388-2392Crossref PubMed Scopus (334) Google Scholar). p62 and p44 are the mammalian counterparts of the yeast TFB1 and SSL1 gene products that are required for DNA nucleotide excision repair (16Wang Z. Buratowski S. Svejstrup J.Q. Feaver W.J. Wu X. Kornberg R.D. Donahue T.F. Friedberg E.C. Mol. Cell. Biol. 1995; 15: 2288-2293Crossref PubMed Scopus (75) Google Scholar). p34 exhibits partial sequence homology to p44 and also contains zinc-finger motifs (17Humbert S. Van Vuuren H. Lutz Y. Hoeijmakers J.H.J. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar). The p40, p37, and p36 subunits of TFIIH are identical to the vertebrate CAK complexes, CDK7, cyclin H, and p36/MAT1, respectively (5Roy R. Adamczewski J.P. Seroz T. Vermuelen W. Tassan J.-P. Schaeffer L. Nigg E.A. Hoeijmakers J.H.J. Egly J.-M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 6Serizawa H. MäKelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar, 7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar). Two subcomplexes containing some TFIIH polypeptides can also be isolated from extracts of HeLa cells (18Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 19Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.-Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar): (i) a five-subunit core TFIIH complex that includes ERCC3 (XPB), p62, p52, p44, and p34 but is devoid of detectable levels of ERCC2 (XPD) or CAK; (ii) an XPD·CAK complex that includes XPD and all three CAK components (CDK7, cyclin H, and p36/MAT1). The addition of XPD·CAK to the core TFIIH potently stimulates the TFIIH transcriptional activity (18Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 19Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.-Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). These observations suggest that core TFIIH and XPD·CAK interact to form a complex that constitutes the TFIIH holoenzyme (holo-TFIIH). Biochemical analysis has therefore revealed that CDK7 is a component of at least three complexes, the trimeric CAK complex (20Devault A. Martinez A.-M. Fesquet D. Labbe J.-C. Morin N. Tassan J.-P. Nigg E.A. Cavadore J.-C. Doree M. EMBO J. 1995; 14: 5027-5036Crossref PubMed Scopus (201) Google Scholar, 21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar, 22Tassan J.-P. Jaquenoud M. Fry A.M. Frutiger S. Hughes G.J. Nigg E.A. EMBO J. 1995; 14: 5608-5617Crossref PubMed Scopus (172) Google Scholar), the quaternary complex with the XPD, and the nine-subunit TFIIH complex (18Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 19Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.-Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). In addition, a number of studies have suggested that the trimeric CAK complex is the CDK7-containing complex involved in cell cycle control (21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar, 23Rossignol M. Kolb-Cheynel I. Egly J.-M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (168) Google Scholar). These studies were based on initial reports that identified the trimeric CAK complex as the kinase responsible for activating a number of cdks (1Fesquet D. Labbe J.-C. Derancourt J. Capony J.-P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.-C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar, 2Poon R.Y.C. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (333) Google Scholar, 3Solomon M.J. Harper J.W. Shuttleworth J. EMBO J. 1993; 12: 3133-3142Crossref PubMed Scopus (273) Google Scholar). However, in Saccharomyces cerevisiae, Kin28 and Ccl1, the counterparts of CDK7 and cyclin H, associate with S. cerevisiae TFIIH, although they do not exhibit detectable CAK activity (4Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 24Cismowski M.J. Laff G.M. Solomon M.J. Reed S.I. Mol. Cell. Biol. 1995; 15: 2983-2992Crossref PubMed Scopus (189) Google Scholar). The CAK activity of S. cerevisiae has recently been identified as the gene product ofCak1/Civ1 (25Kaldis P. Sutton A. Solomon M.J. Cell. 1996; 86: 553-564Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Thuret J.-V. Valay J.-G. Faye G. Mann C. Cell. 1996; 86: 565-576Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, a recent report indicates that CDK7 is essential for mitosis and cdk-activating kinase activity in Drosophila melanogaster (27Larochelle S. Pandur J. Fisher R.P. Salz H.K. Suter B. Genes Dev. 1998; 12: 370-381Crossref PubMed Scopus (150) Google Scholar). Here we show that the trimeric CAK complex exhibits an inhibitory activity in RNAPII-dependent transcription. This inhibition results from the preclusion of TFIIF and RNAPII from the preinitiation complex. These studies reveal a novel role for the trimeric CAK in regulation of transcription. Trimeric CAK was purified from 3 g of HeLa nuclear extract (Fig. 1A). Nuclear extract was loaded on a 1-liter column of phosphocellulose (Whatman) and fractionated stepwise by the indicated KCl concentrations in buffer A (20 mm Tris-HCl, pH 7.9, 0.2 mm EDTA, 10 mm 2-mercaptothanol, 20% glycerol, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. The phosphocellulose 0.3 m KCl fraction (400 mg) was dialyzed to 0.1 m KCl in buffer A and loaded on a 100-ml DEAE-Sephacel column (Amersham Pharmacia Biotech). The column was eluted with 0.5 m KCl in buffer A. The 0.5m KCl elution (260 mg) was dialyzed to 100 mmKCl in buffer A and loaded on a 100-ml Q-Sepharose column (Sigma). The column was resolved using a linear 10-column volume gradient of 100–600 mm KCl. Fractions containing CDK7 (∼200 mm KCl, 37 mg) were dialyzed to 10 mm potassium phosphate in buffer B (5 mm Hepes, pH 7.5, 1 mmdithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 10 μm CaCl2, 10% glycerol, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) and loaded on a 20-ml hydroxyapatite column (American International Chemical). The column was resolved using a linear 10-column volume gradient of 10–600 mm potassium phosphate in buffer B. CDK7-containing fractions (100 mmpotassium phosphate, 8.6 mg) were dialyzed to 1 m ammonium sulfate in buffer C (20 mm Hepes, pH 7.9, 4 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm EDTA, 10% glycerol) and fractionated on a phenyl-Superose HR 5/5 column (Amersham Pharmacia Biotech). The phenyl-Superose column was resolved using a linear 10-column volume gradient of 1 m to 0 mm ammonium sulfate in buffer A. CDK7-containing fractions (∼0.4 m ammonium sulfate, 0.5 mg) were precipitated with 60% ammonium sulfate and fractionated on a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated in 1 m KCl in buffer A. We generated and purified CAK using a detailed protocol for the production of CAK in insect SF9 cells as described previously (21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar). CTD kinase assays for phosphorylation of the carboxyl-terminal domain of RNAPII were described previously (7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar). Transcription assays were reconstituted as described (7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar) using recombinant TBP (10 ng), TFIIB (10 ng), TFIIE (10 ng), TFIIF (10 ng), highly purified HeLa holo-TFIIH (7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar) or highly purified rat TFIIH (6Serizawa H. MäKelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar), and highly purified HeLa core RNAPII (28Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Crossref PubMed Scopus (248) Google Scholar). The rat somatostatin promoter (29Sun P. Schoderbec W.E. Maurer R.A. Mol. Endocrinol. 1992; 11: 1858-1866Google Scholar), human T-cell leukemia virus, type 1 (HTLV-1) promoter (30Kashanchi F. Duvall J.F. Kwok R.P.S. Lundblad J.R. Goodman R.H. Brady J.N. J. Biol. Chem. 1998; 273: 34646-34652Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), or Ad-MLP leading to a 390-nucleotide G-less cassette was used as the template (100 ng). The trimeric CAK complex from HeLa nuclear extract was purified as described under “Experimental Procedures” (Fig. 1A) using the CTD-kinase activity and Western blot analysis with antibodies against CDK7 and cyclin H. Nuclear extract was initially fractionated by phosphocellulose chromatography. The 0.3 m KCl step elution contained approximately 40% of CDK7 immunoreactivity and was devoid of TFIIH activity or ERCC3 immunoreactivity, which fractionated into the 0.5 m KCl step elution (data not shown). The 0.3m KCl fraction was therefore further purified through five additional steps (Fig. 1A). Analysis of the CTD kinase activity of CAK on the sizing column (Superose 6) revealed the peak of activity eluting at ∼200 kDa, consistent with the previously reported size for the trimeric CAK complex (21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar). We estimate that our purification resulted in ∼2000-fold enrichment in CAK. Analysis of column fractions in a transcription system reconstituted with recombinant TBP, TFIIB, TFIIE, TFIIF, highly purified HeLa or rat holo-TFIIH, and highly purified HeLa core RNAPII revealed an inhibitory activity copurifing with the CDK7 complex throughout the purification (Fig. 1B). The transcriptional inhibitory activity was also observed in the last two steps of the purification (phenyl-Superose and Superose 6) (Fig. 1, C and D). Highest concentrations of the CAK complex tested resulted in about 95% inhibition in basal transcription. To determine if the inhibitory activity associated with the HeLa fractions is the CAK complex, we expressed either the trimeric (CDK7-cyclin H-MAT1) or the dimeric CAK complex (CDK7-cyclin H) in insect SF9 cells. These complexes were purified as previously reported (21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar) (Fig. 2A). Moreover, as the highly purified holo-TFIIH complex also contains CAK, we sought to determine the action of the trimeric CAK complex in an assay free of TFIIH. Similar to a previous report (31Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (308) Google Scholar), reconstituted transcription using the rat supercoiled somatostatin promoter is independent of TFIIH or TFIIE but is highly stimulated by these factors (Fig. 2B). Analysis of recombinant trimeric CAK complex in reconstituted transcription assay either in the presence (Fig. 2C, lanes 1 and 2) or the absence (lanes 3–5) of TFIIH revealed that this complex is a potent inhibitor of transcription. The inhibitory effect of CAK is achieved at roughly a 1:1 stoichiometry (0.25 pmol) with other basal factors. To further ascertain the role of the p36/MAT1 subunit of the CDK7 complex in transcriptional repression, we compared the recombinant dimeric and trimeric CAK complexes. Although the two CAK complexes displayed similar kinase activity as ascertained by phosphorylation of a CTD peptide (Fig. 3A), analysis of the dimeric CAK in transcription revealed that p36/MAT1 is required for the inhibitory activity of the CAK complex (Fig. 3B). In contrast to the trimeric CAK, which inhibited transcription driven from either the supercoiled somatostatin (lanes 2 and 3) or supercoiled HTLV-1 (lanes 8 and 9) promoters, the dimeric CAK was devoid of any inhibitory activity with either promoter (lanes 4–6 or 10and 11). These data indicate that the p36/MAT1 subunit of the CAK complex is required for inhibition of transcription and that the CAK-mediated inhibition is independent of the promoter used. To determine whether the p36/MAT1 subunit of the CAK complex is sufficient for transcriptional repression, the p36/MAT1 subunit was expressed inEscherichia coli, and the purified p36/MAT1 was analyzed for its activity in transcription. In contrast to trimeric CAK (Fig. 3B, lane 2), the addition of p36/MAT1 not only failed to inhibit transcription (lanes 3–5) but also displayed a small stimulatory activity (compare lanes 1 and 3). These results demonstrate that although p36/MAT1 is required for the inhibitory activity of the CAK complex, it is not sufficient for inhibition. The p36/MAT1 subunit of CAK was produced in SF9 cells, and purified protein was tested for its ability to confer repression when added to the dimeric CAK complex. As Fig. 3C indicates, neither the dimeric CAK (lane 2) nor the p36/MAT1 protein alone were sufficient to mediate repression. However, the addition of the p36/MAT1 subunit to dimeric CAK reconstitutes the transcriptional repression observed with trimeric CAK (lane 4). To analyze which step during the formation of the preinitiation complex the trimeric CAK may target to repress transcription, we incubated the basal transcription factors in a stepwise fashion with the DNA for 30 min before adding the trimeric CAK complex (Fig. 4A). Preincubation of DNA with either TBP (lane 2) or TBP and TFIIB (lane 3) could not overcome the inhibitory effect of CAK, indicating that the TBP·TFIIB complex (TB) formation is not the target of the CAK complex. However, the addition of TFIIF to the preinitiation complex, which results in the formation of the TBF complex, could partially relieve the CAK repression (lane 4). Formation of the TBPolF or TBPolFE complex by further preincubation with RNAPII or RNAPII and TFIIE resulted in a complete recovery of transcription (lanes 5 and 6). These results indicate that trimeric CAK precludes the entry of RNAPII and TFIIF into a competent preinitiation complex, and a preformed preinitiation complex is refractory to the action of trimeric CAK. This contention is further substantiated when we analyzed whether the addition of excess TFIIF or RNAPII can overcome the inhibitory activity of the trimeric CAK complex. As shown in Fig. 4B, the addition of increasing amounts of TFIIF partially overcomes the CAK-mediated repression (lanes 3 and 4), whereas the addition of excess RNAPII could completely restore transcription (lane 5). We conclude that trimeric CAKs repress transcription by precluding RNAPII and TFIIF entry into the preinitiation complex. To address whether the kinase activity of CDK7 plays a role in CAK-mediated inhibition, we analyzed a kinase-deficient mutant of CDK7, in which lysine 41 was replaced by alanine (Fig. 3A, lane 3). The purified dimeric CAK/K41A, produced in insect cells, was mixed with the p36/MAT1 subunit, produced in insect cells, and analyzed in a reconstituted transcription system. As Fig. 4C indicates, the addition of kinase-deficient CAK resulted in a potent inhibition of transcription (compare lane 1 to lanes 2 and 3). These results indicate that CAK-mediated inhibition is not because of the kinase activity of CAK and may result from CAK physically destabilizing the preinitiation complex. Because TFIIH displays a stimulatory activity in transcription by stabilizing the preinitiation complex, we analyzed whether increasing concentrations of TFIIH can relieve the CAK-mediated repression. As Fig. 4D reveals, the addition of excess TFIIH can overcome the inhibitory activity of CAK (compare lanes 2–4 to 6–8). These results indicate that TFIIH and CAK are in a competition for the preinitiation complex. Whereas TFIIH stimulates transcription by stabilizing the preinitiation complex formation, CAK exerts an inhibitory effect by disrupting its formation. The novelty of this work lies in the following. First, it demonstrates transcriptional inhibitory activity for trimeric CAK in a fully defined system, comprised of essentially homogeneous basal factors and RNA polymerase II. Second, it shows that CAK inhibits transcription by preventing RNA polymerase II and TFIIF entry into the preinitiation complex. Third, it presents evidence for the requirement of p36/MAT1 in the CAK-mediated inhibitory effect. Finally, it demonstrates that the inhibitory activity is independent of the kinase activity of CDK7. The trimeric CAK complex was initially identified as the kinase complex responsible for phosphorylation and consequent activation of other cyclin-dependent kinases from mammalian cells (1Fesquet D. Labbe J.-C. Derancourt J. Capony J.-P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.-C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar, 2Poon R.Y.C. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (333) Google Scholar, 3Solomon M.J. Harper J.W. Shuttleworth J. EMBO J. 1993; 12: 3133-3142Crossref PubMed Scopus (273) Google Scholar). It was later discovered that CDK7, cyclin H, and MAT1 were also components of the basal transcription factor TFIIH (4Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 5Roy R. Adamczewski J.P. Seroz T. Vermuelen W. Tassan J.-P. Schaeffer L. Nigg E.A. Hoeijmakers J.H.J. Egly J.-M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 6Serizawa H. MäKelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar, 7Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar). Furthermore, it was observed that TFIIH can be dissociated into two subcomplexes, one containing the core TFIIH subunits (XPB, p62, p51, p44, and p34) and the other containing XPD and the three CAK subunits (18Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 19Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.-Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). We found that the trimeric CAK purified from HeLa cells contained a transcriptional inhibitory activity in a highly purified reconstituted transcription system. The inhibitory activity associated with the HeLa fractions was demonstrated to be mediated by the trimeric CAK complex, because the recombinant trimeric CAK produced in insect cells inhibited transcription. Interestingly, the p36/MAT1 subunit of the trimeric CAK was required for the transcriptional inhibition. This observation lends further support to the physiological relevance of CAK-mediated inhibition, as the predominant form of CAK in mammalian extracts not associated with TFIIH contains the p36/MAT1 subunit (19Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.-Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar, 21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar). The transcriptional inhibition by the CAK complex did not result from CDK7 kinase activity, because the kinase-deficient mutant of CAK is also a potent inhibitor of transcription. Our studies revealed that CAK inhibited transcription by preventing the formation of the TBPolF complex. Therefore, either preforming the TBPolF complex or the addition of excess TFIIF, RNAPII, or TFIIH was able to stabilize the complex and to overcome the inhibitory effect of CAK. A number of studies have concluded that the trimeric CAK complex represents the form of CAK involved in cell cycle control (1Fesquet D. Labbe J.-C. Derancourt J. Capony J.-P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.-C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar, 2Poon R.Y.C. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (333) Google Scholar, 3Solomon M.J. Harper J.W. Shuttleworth J. EMBO J. 1993; 12: 3133-3142Crossref PubMed Scopus (273) Google Scholar,21Fisher R.P. Methods Enzymol. 1997; 283: 256-270Crossref PubMed Scopus (8) Google Scholar). Here we present evidence indicating that the trimeric CAK complex displays a novel role in transcription distinct from that of its function when associated with TFIIH. Our findings are a further support for CAK as a regulator of transcription in addition to the function of CAK in cell cycle control. We thank Yuying Zhang for expert technical assistance and P. Lieberman for critical comments on the manuscript. We thank the following people for providing reagents used for this study: Y. Xiong for pET-CDK7 and pET-MAT1; R. Roeder for pHT7MAT1; and D. Morgan for baculoviruses carrying CDK7, cyclin H, or p36/MAT1." @default.
• W2019946452 created "2016-06-24" @default.
• W2019946452 creator A5010080958 @default.
• W2019946452 creator A5015281795 @default.
• W2019946452 creator A5024187795 @default.
• W2019946452 creator A5029331756 @default.
• W2019946452 creator A5037825847 @default.
• W2019946452 creator A5085435182 @default.
• W2019946452 date "1999-05-01" @default.
• W2019946452 modified "2023-09-28" @default.
• W2019946452 title "Inhibition of Transcription by the Trimeric Cyclin-dependent Kinase 7 Complex" @default.
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