The Experts below are selected from a list of 501 Experts worldwide ranked by ideXlab platform

Danny Reinberg - One of the best experts on this subject based on the ideXlab platform.

  • mechanism of atp dependent promoter melting by Transcription Factor IIH
    Science, 2000
    Co-Authors: Tae Kyung Kim, Richard H Ebright, Danny Reinberg
    Abstract:

    We show that Transcription Factor IIH ERCC3 subunit, the DNA helicase responsible for adenosine triphosphate (ATP)-dependent promoter melting during Transcription initiation, does not interact with the promoter region that undergoes melting but instead interacts with DNA downstream of this region. We show further that promoter melting does not change protein-DNA interactions upstream of the region that undergoes melting but does change interactions within and downstream of this region. Our results rule out the proposal that IIH functions in promoter melting through a conventional DNA-helicase mechanism. We propose that IIH functions as a molecular wrench: rotating downstream DNA relative to fixed upstream protein-DNA interactions, thereby generating torque on, and melting, the intervening DNA.

  • promoter proximal stalling results from the inability to recruit Transcription Factor IIH to the Transcription complex and is a regulated event
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Prasanna K Kumar, Sasha Akoulitchev, Danny Reinberg
    Abstract:

    Promoter-proximal stalling, a general phenomenon observed during the expression of many RNA polymerase II transcribed genes, is dependent on Transcription Factor IIH (TFIIH). Reactions lacking TFIIH initiated Transcription, but the Transcription complex encountered a block to elongation proximal to the promoter. The accumulation of promoter-proximal stalled complexes was reduced in the presence of TFIIH and efficient escape from this site also required an activator. Promoter-proximal stalled complexes could not be induced to resume elongation. Our results indicate that effective recruitment of TFIIH into Transcription complexes is achieved during formation of the preinitiation complex at the promoter. The studies establish that promoter clearance is a regulated event that requires TFIIH.

  • Immunoaffinity Purification of the Human Multisubunit Transcription Factor IIH
    Journal of Biological Chemistry, 1998
    Co-Authors: Gary Leroy, Lisa Weis, Ronny Drapkin, Danny Reinberg
    Abstract:

    Abstract 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.

  • Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Ronny Drapkin, Sasha Akoulitchev, Danny Reinberg
    Abstract:

    Abstract Transcription Factor IIH (TFIIH) is a multisubunit complex required for Transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH Transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.

  • the 62 and 80 kda subunits of Transcription Factor IIH mediate the interaction with epstein barr virus nuclear protein 2
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Xiao Tong, Ronny Drapkin, Danny Reinberg, Elliott Kieff
    Abstract:

    EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general Transcription Factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted Transcription Factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro Transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Jean Marc Egly - One of the best experts on this subject based on the ideXlab platform.

  • nucleotide excision repair and Transcriptional regulation tfIIH and beyond
    Annual Review of Biochemistry, 2016
    Co-Authors: Emmanuel Compe, Jean Marc Egly
    Abstract:

    Transcription Factor IIH (TFIIH) is a multiprotein complex involved in both Transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this Factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.

  • abnormal xpd induced nuclear receptor transactivation in dna repair disorders trichothiodystrophy and xeroderma pigmentosum
    European Journal of Human Genetics, 2013
    Co-Authors: Xiaolong Zhou, Jean Marc Egly, Emmanuel Compe, Sikandar G Khan, Deborah Tamura, Takahiro Ueda, Jennifer Boyle, John J Digiovanna, Kenneth H Kraemer
    Abstract:

    XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in Transcription as a structural bridge tying the Transcription Factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

  • a role of the c terminal part of p44 in the promoter escape activity of Transcription Factor IIH
    Journal of Biological Chemistry, 2001
    Co-Authors: Alexandre Tremeaubravard, Christophe Perez, Jean Marc Egly
    Abstract:

    Abstract The p44 subunit plays a crucial role in the overall activity of the Transcription/DNA repair Factor TFIIH: on the one hand its N-terminal domain interacts with and regulates the XPD helicase (1, 2); on the other hand, as shown in the present study, it participates with the promoter escape reaction. Mutagenesis along with recombinant technology using the baculovirus/insect cells expression system allowed us to define the function of the two structural motifs of the C-terminal moiety of p44: mutations within the C4 zinc finger motif (residues 291–308) prevent incorporation of the p62 subunit within the core TFIIH. Double mutations in the RING finger motif (residues 345–385) allow the synthesis of the first phosphodiester bond by RNA polymerase II, but prevent its escape from the promoter. This highlights the role of Transcription Factor IIH in the various steps of the Transcription initiation process.

  • A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of Transcription Factor IIH.
    The Journal of biological chemistry, 2001
    Co-Authors: Björn Sandrock, Jean Marc Egly
    Abstract:

    Abstract To understand the role of the various components of TFIIH, a DNA repair/Transcription Factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of Transcription Factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK·XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.

  • mechanism of promoter melting by the xeroderma pigmentosum complementation group b helicase of Transcription Factor IIH revealed by protein dna photo cross linking
    Molecular and Cellular Biology, 2000
    Co-Authors: Maxime Douziech, Jean Marc Egly, Frederic Coin, Jeanmarc Chipoulet, Yoko Arai, Yoshiaki Ohkuma, Benoit Coulombe
    Abstract:

    The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of Transcription Factor IIH (TFIIH) is essential for promoter melting prior to Transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions -9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions -5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

Roger D Kornberg - One of the best experts on this subject based on the ideXlab platform.

  • real time observation of the initiation of rna polymerase ii Transcription
    Nature, 2015
    Co-Authors: Furqan M Fazal, Roger D Kornberg, Cong A Meng, Kenji Murakami, Steven M Block
    Abstract:

    Biochemical and structural studies have shown that the initiation of RNA polymerase II Transcription proceeds in the following stages: assembly of the polymerase with general Transcription Factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a Transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the Transcription Factor IIH involved the rapid opening of an extended Transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

  • revised subunit structure of yeast Transcription Factor IIH tfIIH and reconciliation with human tfIIH
    Journal of Biological Chemistry, 2003
    Co-Authors: Yuichiro Takagi, Hirofumi Komori, Weihau Chang, Andy Hudmon, Hediye Erdjumentbromage, Paul Tempst, Roger D Kornberg
    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.

  • genes for tfb2 tfb3 and tfb4 subunits of yeast Transcription repair Factor IIH homology to human cyclin dependent kinase activating kinase and IIH subunits
    Journal of Biological Chemistry, 1997
    Co-Authors: Errol C. Friedberg, Zhigang Wang, Jesper Q Svejstrup, William J Feaver, Lynn N Henry, David A Bushnell, Roger D Kornberg
    Abstract:

    Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase Transcription Factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit Transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the Factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast Factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

  • the yeast tfb1 and ssl1 genes which encode subunits of Transcription Factor IIH are required for nucleotide excision repair and rna polymerase ii Transcription
    Molecular and Cellular Biology, 1995
    Co-Authors: Zhigang Wang, Jesper Q Svejstrup, William J Feaver, Roger D Kornberg, Stephen Buratowski, T F Donahue, Errol C. Friedberg
    Abstract:

    The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II Transcription Factor TFIIH (Factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II Transcription but are proficient for base excision repair. RNA polymerase II-dependent Transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified Factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal Transcription and NER. Our results also suggest that the repair and Transcription functions of Tfb1 and Ssl1 are separable.

  • rna polymerase Transcription Factor IIH holoenzyme from yeast
    Journal of Biological Chemistry, 1994
    Co-Authors: Jesper Q Svejstrup, William J Feaver, J W Lapointe, Roger D Kornberg
    Abstract:

    An RNA polymerase Transcription Factor IIH holoenzyme (holoTFIIH) has been resolved to near homogeneity from Saccharomyces cerevisiae. HoloTFIIH comprises the five-subunit core Transcription Factor described previously (Feaver, W. J., Svejstrup, J. Q., Bardwell, A. J., Bardwell, L., Buratowski, S., Gulyas, K. D., Donahue, T. F., Friedberg, E. C. and Kornberg, R. D. (1993) Cell 75, 1379-1387) and in addition, SSL2 and three further, as yet unidentified, polypeptides. HoloTFIIH possesses C-terminal repeat domain kinase activity and, together with other pure yeast Transcription proteins, enables RNA polymerase II Transcription in a fully defined system. By contrast, core TFIIH is inert in both C-terminal repeat domain kinase and reconstituted Transcription assays.

Ronny Drapkin - One of the best experts on this subject based on the ideXlab platform.

  • Immunoaffinity Purification of the Human Multisubunit Transcription Factor IIH
    Journal of Biological Chemistry, 1998
    Co-Authors: Gary Leroy, Lisa Weis, Ronny Drapkin, Danny Reinberg
    Abstract:

    Abstract 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.

  • Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Ronny Drapkin, Sasha Akoulitchev, Danny Reinberg
    Abstract:

    Abstract Transcription Factor IIH (TFIIH) is a multisubunit complex required for Transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH Transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.

  • the 62 and 80 kda subunits of Transcription Factor IIH mediate the interaction with epstein barr virus nuclear protein 2
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Xiao Tong, Ronny Drapkin, Danny Reinberg, Elliott Kieff
    Abstract:

    EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general Transcription Factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted Transcription Factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro Transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

  • Cdk-activating kinase complex is a component of human Transcription Factor TFIIH
    Nature, 1995
    Co-Authors: Ramin Shiekhattar, David O Morgan, Robert P Fisher, Fred Mermelstein, Brian David Dynlacht, Holly C. Wessling, Ronny Drapkin, Danny Reinberg
    Abstract:

    Transcription Factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII)1–3. Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent Transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between Transcription and the cell cycle machinery.

William J Feaver - One of the best experts on this subject based on the ideXlab platform.

  • genes for tfb2 tfb3 and tfb4 subunits of yeast Transcription repair Factor IIH homology to human cyclin dependent kinase activating kinase and IIH subunits
    Journal of Biological Chemistry, 1997
    Co-Authors: Errol C. Friedberg, Zhigang Wang, Jesper Q Svejstrup, William J Feaver, Lynn N Henry, David A Bushnell, Roger D Kornberg
    Abstract:

    Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase Transcription Factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit Transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the Factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast Factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

  • the yeast tfb1 and ssl1 genes which encode subunits of Transcription Factor IIH are required for nucleotide excision repair and rna polymerase ii Transcription
    Molecular and Cellular Biology, 1995
    Co-Authors: Zhigang Wang, Jesper Q Svejstrup, William J Feaver, Roger D Kornberg, Stephen Buratowski, T F Donahue, Errol C. Friedberg
    Abstract:

    The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II Transcription Factor TFIIH (Factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II Transcription but are proficient for base excision repair. RNA polymerase II-dependent Transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified Factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal Transcription and NER. Our results also suggest that the repair and Transcription functions of Tfb1 and Ssl1 are separable.

  • rna polymerase Transcription Factor IIH holoenzyme from yeast
    Journal of Biological Chemistry, 1994
    Co-Authors: Jesper Q Svejstrup, William J Feaver, J W Lapointe, Roger D Kornberg
    Abstract:

    An RNA polymerase Transcription Factor IIH holoenzyme (holoTFIIH) has been resolved to near homogeneity from Saccharomyces cerevisiae. HoloTFIIH comprises the five-subunit core Transcription Factor described previously (Feaver, W. J., Svejstrup, J. Q., Bardwell, A. J., Bardwell, L., Buratowski, S., Gulyas, K. D., Donahue, T. F., Friedberg, E. C. and Kornberg, R. D. (1993) Cell 75, 1379-1387) and in addition, SSL2 and three further, as yet unidentified, polypeptides. HoloTFIIH possesses C-terminal repeat domain kinase activity and, together with other pure yeast Transcription proteins, enables RNA polymerase II Transcription in a fully defined system. By contrast, core TFIIH is inert in both C-terminal repeat domain kinase and reconstituted Transcription assays.