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• W2018133293 abstract "Tfb4 is identified as a subunit of the core complex of yeast RNA polymerase II general transcription factor IIH (TFIIH) by affinity purification, by peptide sequence analysis, and by expression of the entire complex in insect cells. Tfb3, previously identified as a component of the core complex, is shown instead to form a complex with cdk and cyclin subunits of TFIIH. This reassignment of subunits resolves a longstanding discrepancy between yeast and human TFIIH complexes. Tfb4 is identified as a subunit of the core complex of yeast RNA polymerase II general transcription factor IIH (TFIIH) by affinity purification, by peptide sequence analysis, and by expression of the entire complex in insect cells. Tfb3, previously identified as a component of the core complex, is shown instead to form a complex with cdk and cyclin subunits of TFIIH. This reassignment of subunits resolves a longstanding discrepancy between yeast and human TFIIH complexes. TFIIH 1The abbreviations used are: TF, transcription factor; GST, gluthathione S-transferase; TAP, tandem affinity purification; TEV, tobacco etch potyvirus; Ni-NTA, Ni2+-nitrilotriacetic acid-agarose; pol II, polymerase II; cdk, cyclin-dependent protein kinase; ORF, open reading frame; MALDI-reTOF, matrix-assisted laser-desorption/ionization reflectron time-of-flight; MS, mass spectrometry; CBP, calmodulin-binding peptide.1The abbreviations used are: TF, transcription factor; GST, gluthathione S-transferase; TAP, tandem affinity purification; TEV, tobacco etch potyvirus; Ni-NTA, Ni2+-nitrilotriacetic acid-agarose; pol II, polymerase II; cdk, cyclin-dependent protein kinase; ORF, open reading frame; MALDI-reTOF, matrix-assisted laser-desorption/ionization reflectron time-of-flight; MS, mass spectrometry; CBP, calmodulin-binding peptide. is remarkable among RNA polymerase II (pol II) transcription factors for its size, catalytic activities, and multiple functional roles (1Svejstrup J.Q. Vichi P. Egly J.M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Consisting of nine subunits, with a total mass of about 500 kDa, TFIIH is comparable in size and complexity to pol II. The largest subunits of TFIIH, termed Ssl2 and Rad3 in yeast, are DNA-dependent ATPase/helicases and are essential for unwinding promoter DNA at the active center of pol II (2Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H. Chambon P. Egly J.M. Science. 1993; 260: 58-63Crossref PubMed Scopus (665) Google Scholar, 3Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 4Guzder S.N. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (160) Google Scholar, 5Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar, 6Sung P. Prakash L. Weber S. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6045-6049Crossref PubMed Scopus (90) Google Scholar, 7Sung P. Bailly V. Weber C. Thompson L.H. Prakash L. Prakash S. Nature. 1993; 365: 852-855Crossref PubMed Scopus (284) Google Scholar, 8Bootsma D. Hoeijmakers J.H. Nature. 1993; 363: 114-115Crossref PubMed Scopus (199) Google Scholar, 9Schaeffer L. Moncollin V. Roy R. Staub A. Mezzina M. Sarasin A. Weeda G. Hoeijmakers J.H. Egly J.M. EMBO J. 1994; 13: 2388-2392Crossref PubMed Scopus (333) Google Scholar). Two smaller subunits form a cyclin-dependent protein kinase (cdk)-cyclin pair that phosphorylates the C-terminal domain of pol II during the transition from transcription initiation to elongation (10Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (359) Google Scholar, 11Lu H. Zawel L. Fisher L. Egly J.M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar). Beyond its role in transcription, six TFIIH subunits, including Ssl2 and Rad3, are components of a DNA “repairosome,” responsible for nucleotide excision repair of DNA damage (12Svejstrup J.Q. Wang Z. Feaver W.J. Wu X. Bushnell D.A. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1995; 80: 21-28Abstract Full Text PDF PubMed Scopus (239) Google Scholar, 13Feaver W.J. Huang W. Gileadi O. Myers L. Gustafsson C.M. Kornberg R.D. Friedberg E.C. J. Biol. Chem. 2000; 275: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In human cells, the counterparts of the cdk-cyclin pair perform yet another role, activating the cdks that drive the cell cycle (14Devault 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 (200) Google Scholar, 15Tassan J.P. Schultz S.J. Bartek J. Nigg E.A. J. Cell Biol. 1994; 127: 467-478Crossref PubMed Scopus (216) Google Scholar). All nine subunits of TFIIH have been conserved in amino acid sequence from yeast to humans (1Svejstrup J.Q. Vichi P. Egly J.M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar), and structural studies have demonstrated conservation as well (16Schultz P. Fribourg S. Poterszman A. Mallouh V. Moras D. Egly J.M. Cell. 2000; 102: 599-607Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17Chang W.H. Kornberg R.D. Cell. 2000; 102: 609-613Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). An intact nine-subunit “holo” TFIIH, capable of fulfilling the requirement for transcription, has been isolated from both yeast (18Svejstrup J.Q. Feaver W.J. LaPointe J. Kornberg R.D. J. Biol. Chem. 1994; 269: 28044-28048Abstract Full Text PDF PubMed Google Scholar) and mammalian cells (19Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7356-7360Crossref PubMed Scopus (92) Google Scholar, 20Gerard 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, 21Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Abstract Full Text PDF PubMed Google Scholar). Subcomplexes, apparently related to the distinct functional roles of TFIIH, have been reported (22Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In previous work from this laboratory, a 5-subunit “core” complex of the yeast proteins Rad3, Ssl1, Tfb1, Tfb2, and Tfb3 was described (18Svejstrup J.Q. Feaver W.J. LaPointe J. Kornberg R.D. J. Biol. Chem. 1994; 269: 28044-28048Abstract Full Text PDF PubMed Google Scholar, 24Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), as was a separate complex of the cdk-cyclin pair, termed TFIIK (10Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (359) Google Scholar). While subcomplexes of TFIIH subunits were also obtained from human cells (20Gerard 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, 21Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Abstract Full Text PDF PubMed Google Scholar, 25Adamczewski J.P. Rossignol M. Tassan J.P. Nigg E.A. Moncollin V. Egly J.M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar), with similar compositions to those from yeast, a notable discrepancy arose in regard to MAT1, the human counterpart of Tfb3 (25Adamczewski J.P. Rossignol M. Tassan J.P. Nigg E.A. Moncollin V. Egly J.M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar). MAT1 was isolated in association with the cdk-cyclin pair, rather than as part of the core complex (14Devault 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 (200) Google Scholar, 15Tassan J.P. Schultz S.J. Bartek J. Nigg E.A. J. Cell Biol. 1994; 127: 467-478Crossref PubMed Scopus (216) Google Scholar, 25Adamczewski J.P. Rossignol M. Tassan J.P. Nigg E.A. Moncollin V. Egly J.M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar). Another protein, p34, replaced MAT1 in the human core complex (23Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 25Adamczewski J.P. Rossignol M. Tassan J.P. Nigg E.A. Moncollin V. Egly J.M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar, 26Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar). The yeast homolog of p34, termed Tfb4, was late to be identified (24Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and although it was shown to be required for both transcription and nucleotide excision repair (27Feaver W.J. Huang W. Friedberg E.C. J. Biol. Chem. 1999; 274: 29564-29567Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), it was not definitively assigned to a subcomplex. Recent studies (28Keogh M.C. Cho E.J. Podolny V. Buratowski S. Mol. Cell. Biol. 2002; 22: 1288-1297Crossref PubMed Scopus (61) Google Scholar) have indicated an association of Tfb3 with the cdk-cyclin pair rather than with the yeast core complex. We now find that the component of the core complex originally identified as Tfb3 is, in fact, Tfb4. We confirm and extend the evidence for a Tfb3-cdk-cyclin trimer. The revised molecular description of yeast TFIIH is entirely coincident with that of the human factor. Construction of Baculoviruses Containing Genes for TFIIH Subunits and Expression in Insect Cells—Open reading frames (ORFs) of genes encoding, Rad3, Tfb1, Tfb2, Ssl1, Tfb3, and Tfb4, were amplified from yeast genomic DNA by polymerase chain reaction (PCR) and cloned into the BacPAK9 baculovirus expression vector (Clontech). A hexahistidine tag was added at the C terminus of the Tfb1 ORF. Recombinant viruses were produced in monolayer of Sf21 cells as described (Clontech). For protein expression, Sf21 cells (∼1.5 × 107) in a T75 flask were infected with various combinations of cloned virus stocks at a multiplicity of infection of 2–10. After 72 h, the cells were harvested and stored at –80 °C until use. Cells were lysed in 1 ml of buffer A (50 mm Hepes-KOH (pH 7.6), 10% glycerol, and 5 mm β-mercaptoethanol) containing 600 mm potassium acetate, 0.5% Nonidet P-40 (Calbiochem), and protease inhibitor mix (final concentrations of 6 μm leupeptin, 20 μm pepstatin A, 20 μm benzamidine, and 10 μm phenylmethylsulfonyl fluoride). The cell lysate was clarified by centrifugation at 20,000 × g for 30 min and loaded on a 0.5 ml column of Ni-NTA resin (Qiagen) equilibrated with buffer A containing 600 mm potassium acetate and 0.01% Nonidet P-40. After washing with 5 ml of buffer A containing 1.2 m potassium acetate and 0.01% Nonidet P-40, and 5 ml of buffer A containing 150 mm potassium acetate, proteins were eluted with buffer A containing 150 mm potassium acetate and 300 mm imidazole (pH 8.0). Peak fractions (10 μl) were subjected to immunoblot analysis. Construction of GST Fusion Proteins and Antibody Production—The ORFs of yeast SSL1 and TFB4 genes were amplified by PCR and cloned between BamHI and XhoI sites of pGEX6P-1 (Amersham Bioscience). GST-Ssl1 and GST-Tfb4 were overexpressed in Escherichia coli BL21 CodonPlus cells (Strategene), grown in 500 ml of Luria broth at 37 °C to an A 600 value of 0.6–0.8, and induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside for 6 h at room temperature. The cells were harvested, frozen in liquid nitrogen, and ground in a mortar and pestle under liquid nitrogen to a fine powder (5–10 min). After the cell powder was thawed, 50 ml of lysis buffer (phosphate-buffered saline containing 1 m NaCl, 10 mm dithiothreitol, and protease inhibitor mix) was added, followed by stirring for 20 min at 4 °C, brief sonication, and centrifugation at 100,000 × g for 60 min. The supernatant was loaded on a 3 ml column of GST-agarose (Sigma) equilibrated with lysis buffer. The column was washed with 10 volumes of lysis buffer, followed by 10 volumes of lysis buffer without NaCl. GST fusion proteins were eluted with lysis buffer containing 10 mm glutathione and no NaCl. GST-Tfb4 was dialyzed overnight against phosphate-buffered saline, followed by concentration to 2 mg/ml in a Vivaspin 6m concentrator, 30,000 molecular weight cutoff (Vivascience). About 1 mg of GST-Tfb4 was used to inoculate a rabbit (Covance, PA). GST-Ssl1 was fractionated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. The GST-Ssl1 protein band was excised and used to inoculate rabbits (Covance, PA). Construction of TAP-tagged Yeast Strains—Following the published protocol (29Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2280) Google Scholar), PCR was performed with pBS1458 as template and with primer sets targeting either TFB3 or TFB4 genomic loci. The PCR products were used to transform yeast strain CB010 (MATa, pep4::His, prb::Leu, prc1::HISG, can1, ade2, trp1, ura3, leu2–3, 112), yielding the yeast strains YT062 (TFB3-tagged) and YT063 (TFB4-tagged). Affinity Purification of TFIIH Complexes—Yeast strain YT062 or YT063 was grown in 7.5 liter of 2× YPD (4% (w/v) Bacto Peptone, 2% (w/v) yeast extract, 4% (w/v) glucose) to an A 600 value of 8–9. Cells were harvested, wash once with cold water, and the resulting cell pellets (∼180 g) were extruded into liquid nitrogen through a 60-ml syringe. The frozen cells were broken in liquid nitrogen essentially as described previously (30Boeger H. Griesenbeck J. Strattan J.S. Kornberg R.D. Mol. Cell. 2003; 11: 1587-1598Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), using a 2-liter Waring blender at high speed for 10 min with constant addition of liquid nitrogen. About 160 g of broken cells were thawed at 4 °C and 230 ml of 0.27 m Tris acetate (pH 7.6), 1 m ammonium sulfate, 0.09 m potassium acetate, 1.8 mm EDTA, 18% glycerol, 10 mm β-mercaptoethanol, protease inhibitor mix was added. The mixture was stirred at 4 °C for 30 min and clarified by centrifugation in a Beckman JA14 rotor at 13,000 rpm for 20 min and then in a Beckman Ti45 rotor at 42,000 rpm for 90 min. Ammonium sulfate was added to 60% of saturation, followed by centrifugation in a Beckman JA14 rotor at 13, 000 rpm for 45 min. The pellet was resuspended in 50 ml of buffer A (50 mm Hepes-KOH (pH 7.6), 10% glycerol, 5 mm β-mercaptoethanol) containing protease inhibitor mix, clarified by centrifugation in a Beckman Ti45 rotor at 40,000 rpm for 30 min, and loaded on a 0.8 ml IgG-agarose column (Sigma) equilibrated in buffer A containing 500 mm ammonium sulfate at 4 °C. The column was washed with 10 ml of buffer A containing 500 mm ammonium sulfate and with 10 ml of the buffer A containing 100 mm ammonium sulfate. The column was equilibrated with 50 mm Hepes-KOH (pH 8.0), 0.1 mm EDTA, 200 mm potassium acetate, 5 mm β-mercaptoethanol and eluted by incubation overnight in the same buffer containing TEV protease (40 μg/ml) at 4 °C. Peptide Sequence Analysis and Protein Identification—A phenyl column fraction of highly purified core TFIIH (60 μl), prepared as described previously (17Chang W.H. Kornberg R.D. Cell. 2000; 102: 609-613Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), was precipitated with 20% acetone in the cold and subjected to 10% SDS-PAGE. The lowest molecular weight band, visualized with Coomassie Brilliant Blue R-250, was excised. The gel slice was dried in a SpeedVac. The protein was digested with trypsin, peptides were fractionated on a Poros 50 R2 RP micro-tip, and the resulting peptide pools were analyzed by matrix-assisted laser-desorption/ionization reflectron time-of-flight (MALDI-reTOF) MS using a Bruker UltraFlex TOF/TOF instrument (Bruker Daltonics; Bremen, Germany), as described previously (31Erdjument-Bromage H. Lui M. Lacomis L. Grewal A. Annan R.S. McNulty D.E. Carr S.A. Tempst P. J. Chromatogr. Sect. A. 1998; 826: 167-181Crossref PubMed Scopus (195) Google Scholar, 32Sebastiaan Winkler G. Lacomis L. Philip J. Erdjument-Bromage H. Svejstrup J.Q. Tempst P. Methods. 2002; 26: 260-269Crossref PubMed Scopus (70) Google Scholar). Selected experimental masses (m/z) were then taken to search a non-redundant protein data base (∼1.4 × 106 entries; National Center for Biotechnology Information, Bethesda, MD), utilizing the PeptideSearch (Matthias Mann, Southern Denmark University, Odense, Denmark) algorithm. A molecular weight range twice the predicted weight was covered, with a mass accuracy restriction better than 40 ppm and maximum one missed cleavage site allowed per peptide. Mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, using the UltraFlex instrument in “LIFT” mode. Fragment ion spectra were then taken to search the non-redundant protein data base using the MASCOT MS/MS Ion Search program (Matrix Science Ltd., London, UK). Any identification thus obtained was verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data. Immunoblot Analysis—Eluates (10 μl) of Ni-NTA or IgG columns were subjected to 10% SDS-PAGE, transferred to Protran membranes (Schleicher & Schuell), and probed with anti-Rad3, anti-Ssl1, anti-Tfb1, anti-Tfb2, anti-Tfb3, anti-Tfb4, anti-Ccl1, and anti-Kin28 antibodies. Detection was performed either with horseradish peroxidase-conjugated anti-rabbit antibodies (Bio-Rad) followed by ECL (Amersham Biosciences) or with alkaline phosphatase-conjugated anti-rabbit antibodies (Bio-Rad) followed by color development by the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid substrate system (Sigma). Tfb4, but Not Tfb3, Supports Expression of Core TFIIH in Insect Cells—We set out to express yeast TFIIH in insect cells, beginning with the previously defined core complex of Rad3, Ssl1, Tfb1, Tfb2, and Tfb3. A monolayer of Sf21 cells was infected with a mixture of baculoviruses, each harboring a gene for one of the five proteins, with that for Tfb1 bearing a hexahistidine tag. Clarified cell extracts were applied to Ni-NTA resin and eluted with imidazole, and eluted proteins were detected by immunoblotting with antibodies against the five proteins. Only Rad3, Tfb1, and Ssl1 were detected in the eluate (Fig. 1, lane 3). When the experiment was repeated, substituting a virus expressing Tfb4 for that expressing Tfb3, a five-subunit complex was obtained, as shown by the detection of all expressed proteins in the eluate (Fig. 1, lane 6). In control experiments, none of the antibodies showed significant cross-reactivity with proteins in an extract from uninfected cells, and neither Tfb3 nor Tfb4, expressed individually, was retained nonspecifically on Ni-NTA resin (Fig. 1, lanes 9 and 12). We conclude that Tfb4 is required for the assembly of a five-protein core complex, which does not include Tfb3. Peptide Sequence Determination Identifies the Smallest Subunit of Highly Purified Core TFIIH as TFB4 —The assembly of a core complex in insect cells with Tfb4 but not Tfb3 led us to question the previous assignment of the lowest molecular weight band in SDS gels of yeast core TFIIH preparations to Tfb3. As this assignment was based on mass spectroscopy of tryptic peptides derived from the gel band (24Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), we repeated the analysis. We used a more recent, improved yeast core TFIIH preparation of sufficient purity for crystallization (17Chang W.H. Kornberg R.D. Cell. 2000; 102: 609-613Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Following SDS-PAGE (Fig. 2A), the smallest band, of about 37 kDa, was excised, dried, and subjected to MALDI analysis. All peptide fragments detected had sequences derived from Tfb4 (Fig. 2B). We conclude that the smallest subunit of core TFIIH is Tfb4 and that the previous results arose from contamination by Tfb3, nearly identical in size to Tfb4. Affinity Purification of Tfb4 from Yeast Yields Core TFIIH, whereas Affinity Purification of Tfb3 Yields a cdk-cyclin-Tfb3 Complex—Despite the requirement for Tfb4 for assembly of a core TFIIH complex in insect cells, and despite the presence of Tfb4 along with four other proteins in a core TFIIH preparation from yeast, it still remains to be shown that all components of the core preparation are physically associated with one another. To this end, we expressed Tfb4 bearing a TAP tag in yeast. A crude extract was applied to an IgG column for binding the protein A moiety of the TAP tag, and specifically bound proteins were eluted by cleavage of the tag with TEV protease. SDS-PAGE and Coomassie Blue staining showed (Fig. 3A), and immunoblotting confirmed (Fig. 3B) the enrichment of Rad3, Ssl1, Tfb1, and Tfb2, together with Tfb4 (bearing the residual CBP component of the TAP tag). After this single step of affinity purification, only a few impurities remained. When the alternative experiment was performed of expressing TAP-tagged Tfb3 rather than Tfb4, the eluate from the IgG column contained no core TFIIH subunits (Fig. 3). Rather, what emerged were apparently stoichiometric amounts of Tfb3 and the cdk-cyclin pair (Kin28 and Ccl1). Similar evidence for a Tfb3-cdk-cyclin complex has been reported elsewhere (28Keogh M.C. Cho E.J. Podolny V. Buratowski S. Mol. Cell. Biol. 2002; 22: 1288-1297Crossref PubMed Scopus (61) Google Scholar). The results of affinity purification therefore support those from peptide sequence analysis and expression in insect cells, showing that Tfb4 is a subunit of core TFIIH, whereas Tfb3 is not. The results further demonstrate the association of Tfb3 with the cdk-cyclin pair, previously denoted TFIIK (10Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (359) Google Scholar). With this realignment of subunits between core TFIIH and TFIIK, the compositions of the yeast complexes now correspond perfectly with their counterparts in human cells (Table I).Table IYeast TFIIH subunits and their human homologs We thank Drs. P. Fischhaber and E. C. Friedberg for anti-Rad3 antibody, L. Lacomis for help with mass spectrometry, and Dr. J. Griesenbeck for help preparing whole cell extracts." @default.
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• W2018133293 title "Revised Subunit Structure of Yeast Transcription Factor IIH (TFIIH) and Reconciliation with Human TFIIH" @default.
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