The Experts below are selected from a list of 753 Experts worldwide ranked by ideXlab platform

Edith H Wang - One of the best experts on this subject based on the ideXlab platform.

  • Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy
    Nature Publishing Group, 2018
    Co-Authors: Elizabeth C Curran, Hui Wang, Thomas R Hinds, Ning Zheng, Edith H Wang
    Abstract:

    Abstract The general Transcription Factor IID (TFIID) is the first component of the preinitiation complex (PIC) to bind the core promoter of RNA polymerase II transcribed genes. Despite its critical role in protein-encoded gene expression, how TFIID engages promoter DNA remains elusive. We have previously revealed a winged-helix DNA-binding domain in the N-terminal region of the largest TFIID subunit, TAF1. Here, we report the identification of a second DNA-binding module in the C-terminal half of human TAF1, which is encoded by a previously uncharacterized conserved zinc knuckle domain. We show that the TAF1 zinc knuckle aids in the recruit of TFIID to endogenous promoters vital for cellular proliferation. Mutation of the TAF1 zinc knuckle with defects in DNA binding compromises promoter occupancy of TFIID, which leads to a decrease in Transcription and cell viability. Together, our studies provide a foundation to understand how TAF1 plays a central role in TFIID promoter binding and regulation of Transcription initiation

  • Crystal structure of a TAF1-TAF7 complex in human Transcription Factor IID reveals a promoter binding module
    Cell Research, 2014
    Co-Authors: Hui Wang, Elizabeth C Curran, Thomas R Hinds, Edith H Wang, Ning Zheng
    Abstract:

    The general Transcription Factor IID (TFIID) initiates RNA polymerase II-mediated eukaryotic Transcription by nucleating pre-initiation complex formation at the core promoter of protein-encoding genes. TAF1, the largest integral subunit of TFIID, contains an evolutionarily conserved yet poorly characterized central core domain, whose specific mutation disrupts cell proliferation in the temperature-sensitive mutant hamster cell line ts 13. Although the impaired TAF1 function in the ts 13 mutant has been associated with defective Transcriptional regulation of cell cycle genes, the mechanism by which TAF1 mediates Transcription as part of TFIID remains unclear. Here, we present the crystal structure of the human TAF1 central core domain in complex with another conserved TFIID subunit, TAF7, which biochemically solubilizes TAF1. The TAF1-TAF7 complex displays an inter-digitated compact architecture, featuring an unexpected TAF1 winged helix (WH) domain mounted on top of a heterodimeric triple barrel. The single TAF1 residue altered in the ts 13 mutant is buried at the junction of these two structural domains. We show that the TAF1 WH domain has intrinsic DNA-binding activity, which depends on characteristic residues that are commonly used by WH fold proteins for interacting with DNA. Importantly, mutations of these residues not only compromise DNA binding by TAF1, but also abrogate its ability to rescue the ts 13 mutant phenotype. Together, our results resolve the structural organization of the TAF1-TAF7 module in TFIID and unveil a critical promoter-binding function of TAF1 in Transcription regulation.

  • Transcription Factor IID Recruitment and Sp1 Activation DUAL FUNCTION OF TAF1 IN CYCLIN D1 Transcription
    Journal of Biological Chemistry, 2003
    Co-Authors: Traci L. Hilton, Edith H Wang
    Abstract:

    Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the Transcription Factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing Transcription of cell cycle regulators, including cyclin D1. Here we provide evidence that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, we have identified protein-DNA interactions within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which we termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. Our finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated Transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter.

  • Requirement for TAF(II)250 acetyltransferase activity in cell cycle progression.
    Molecular and cellular biology, 2000
    Co-Authors: Elizabeth L. Dunphy, Theron Johnson, Scott S. Auerbach, Edith H Wang
    Abstract:

    The TATA-binding protein (TBP)-associated Factor TAF(II)250 is the largest component of the basal Transcription Factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAF(II)250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G(1). At the nonpermissive temperature (39.5 degrees C), Transcription from only a subset of protein encoding genes, including the G(1) cyclins, is dramatically reduced in the mutant cells. Here we demonstrate that the ability of the ts13 allele of TAF(II)250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5 degrees C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAF(II)250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAF(II)150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or Transcriptional defect observed at 39.5 degrees C. These data suggest that TAF(II)250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes.

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

  • New core promoter element in RNA polymerase II-dependent Transcription: sequence-specific DNA binding by Transcription Factor IIB
    Genes & development, 1998
    Co-Authors: Thierry Lagrange, Danny Reinberg, Achillefs N. Kapanidis, Hong Tang, Richard H. Ebright
    Abstract:

    A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of Transcription Factor IIB to enter Transcription complexes and support Transcription initiation. The sequence element is recognized directly by the Transcription Factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that Transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general Transcription Factor, IIB, in addition to Transcription Factor IID.

  • Purification of human RNA polymerase II and general Transcription Factors
    Methods in enzymology, 1996
    Co-Authors: Edio Maldonado, Ronny Drapkin, Danny Reinberg
    Abstract:

    Publisher Summary The identification and isolation of the general Transcription Factors (GTFs) that govern the activity of the RNA polymerase II (RNAP II) enzyme have paved the way toward understanding gene expression at the molecular level. Conventional biochemical purification schemes have identified five GTFs specifically required for RNAP II-driven basal Transcription. These include Transcription Factor IID (TFIID), which is composed of an array of proteins, called TATA-binding protein-associated Factors (TAFs), and tightly bound to the TATA-binding protein (TBP): TFIIB, TFIIE, TFIIF, and TFIIH. The purification of these GTFs has resulted in the isolation of the complementary DNAs (cDNA) for almost all the Factors required to assemble the basal Transcription apparatus. Recombinant DNA technology has provided convenient methods by which to express and purify the assortment of GTF cDNAs, and reconstitute functionally active Factors. The ability to reconstitute a highly purified Transcription system in vitro enables to probe the role of specific repressors and activators/coactivators in regulating RNAP II Transcription. The chapter highlights purification protocols for the various recombinant and native GTFs and RNAP II from humans.

  • The initiator directs the assembly of a Transcription Factor IID-dependent Transcription complex
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Juan M. Cárcamo, L Buckbinder, Danny Reinberg
    Abstract:

    Abstract Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general Transcription Factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient Transcription utilizing a discrete start site. Complexes formed at either site required Transcription Factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for Transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.

  • Interaction of CAP sequence site binding Factor and Transcription Factor IID preceding and following binding to the adenovirus 2 major late promoter.
    The Journal of biological chemistry, 1991
    Co-Authors: Brian Safer, Danny Reinberg, W F Jacob, E. Maldonado, J. Carcamo, S. Garfinkel, R. Cohen
    Abstract:

    Interaction of cloned yeast, drosophila, and human Transcription Factor IID (yTFIID, dTFIID, and hTFIID, respectively) with the adenovirus 2 major late promoter (Ad2 MLP) confers a more limited pattern of DNase I protection than that obtained using highly purified native hTFIID (Hahn, S., Buratowski, S., Sharp, P. A. and Guarente, L. (1989) EMBO J. 8, 3379-3382; Van Dyke, M. W., and Sawadogo, M. (1990) Mol. Cell. Biol. 10, 3415-3420; Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G. (1989) Nature 341, 299-303; Peterson, M. G., Tanese, N., Pugh, B.F., and Tjian, R. (1990) Science 248, 1625-1630; Hoey, T., Dynlacht, B. D., Peterson, M.G., Pugh, B.F., and Tjian, R. (1990) Cell 61, 1179-1186). Since the mass of the cloned TFIIDs is considerably less than that of native hTFIID (27-38 kDa versus 120-140 kDa), it is considered likely that native hTFIID exists as a mixed heterodimer. We have recently identified, purified, and characterized a novel Transcription Factor that binds to the CAP site region (+1 to +23) of the Ad2 MLP. This CAP site binding Factor, designated CBF, is required for optimal Transcriptional activity. We now show that when bound to the Ad2 MLP, yTFIID and CBF interact to generate the extended pattern of DNase I protection conferred by native hTFIID. In addition, bound yTFIID and CBF interact such that the stability of the complex exceeds that of each Factor bound alone. We also demonstrate the existence in nuclear extracts of a hTFIID and CBF heterodimer by the electrophoretic mobility shift analysis. CBF, therefore, may represent the first identified member of a large family of gene-specific TFIID-associated Factors that are required for the regulated gene-specific expression of TFIID activity.

  • Direct interaction between adenovirus E1A protein and the TATA box binding Transcription Factor IID.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Nobuo Horikoshi, Danny Reinberg, Kathleen Maguire, Anastasia Kralli, Edio Maldonado, Roberto Weinmann
    Abstract:

    Adenovirus E1A has long been known to activate/repress cellular and viral Transcription. The Transcriptional activity of nuclear extracts was depleted after chromatography on immobilized E1A protein columns that specifically retained the Transcription Factor (TF) IID. Stronger direct interactions between E1A and human TFIID than between E1A and yeast TFIID suggest that the unique sequences of the human protein may be involved. We have demonstrated that this interaction occurs directly between bacterially produced E1A and bacterially produced human TFIID in a protein blot assay. We propose that E1A protein may transduce regulatory signals from upstream activators to basal elements of the Transcriptional machinery by contacting TFIID.

Stephen T. Smale - One of the best experts on this subject based on the ideXlab platform.

  • Magnesium-Agarose Electrophoretic Mobility Shift Assay (EMSA) of Transcription Factor IID Binding to DNA
    Cold Spring Harbor protocols, 2010
    Co-Authors: Michael Carey, Craig L. Peterson, Stephen T. Smale
    Abstract:

    The general Transcription Factor IID (TFIID) is a key target for regulation because its binding to a core promoter is the nucleating step in Transcription complex assembly. Many eukaryotic activators stimulate recruitment of the TFIID when its concentration is made limiting at a promoter in vitro. Magnesium-agarose gels can separate large complexes containing TFIID, TFIIA (the DA complex), and TFIIB (the DAB complex) and permit a quantitative measurement of how activators stimulate assembly of such complexes. The advantage of the electrophoretic mobility shift assay (EMSA) is that the reactions can be performed under subsaturating conditions where a TFIID footprint might not be observed. Typically, the activator is incubated with a 32P-labeled DNA template, recombinant TFIIA purified from Escherichia coli, and immunopurified TFIID. After incubation, the samples are electrophoresed on magnesium-containing agarose gels, dried onto DEAE-cellulose paper, and autoradiographed. The DNA-protein complexes containing TFIID migrate with reduced mobility on magnesium-agarose gels both because of the large size of the complex and because the TATA-binding protein (TBP) subunit induces a sharp bend in the DNA, causing altered mobility. By comparing the binding of TFIID over a wide concentration range, with and without activator, one can assess whether the activator interacts with TBP or with one of the TBP-associated Factors (TAFIIs). Additional Factors such as TFIIA and TFIIB can be added subsequently to quantify their contributions to assembly of the Transcription complex.

  • Purification of epitope-tagged Transcription Factor IID.
    Cold Spring Harbor protocols, 2010
    Co-Authors: Michael Carey, Craig L. Peterson, Stephen T. Smale
    Abstract:

    INTRODUCTION Transcription Factor IID (TFIID) is one of the most critical Factors in Transcription complex assembly because it recognizes a core promoter and interacts with chromatin and activator proteins. This protocol uses immunoaffinity chromatography in a simple two-step procedure to purify modified TFIID to homogeneity with limited loss of activity. In brief, a short peptide containing the influenza virus hemagglutinin (HA) tag is fused onto the amino terminus of TATA-binding protein (TBP), and a retroviral transfer system is used to generate a HeLa cell line stably expressing the HA-tagged TBP. Extracts from this cell line contain TFIID, which stably incorporates the epitope-tagged TBP. The TFIID is partially purified from these extracts using phosphocellulose chromatography and then immunopurified using a resin containing protein A-Sepharose beads cross-linked to a monoclonal antibody against the influenza epitope. The TFIID is then eluted from the immunoaffinity resin in pure form using an HA peptide. The resulting TFIID contains a complete complement of TBP-associated Factors (TAFs) and can be used in Transcription, electrophoretic mobility shift assays (EMSA), and footprinting assays; its purity is well suited for many other studies.

  • Sp1 activation of a TATA-less promoter requires a species-specific interaction involving Transcription Factor IID
    Nucleic acids research, 1998
    Co-Authors: Katayoon H. Emami, Thomas W. Burke, Stephen T. Smale
    Abstract:

    Sp1 is a ubiquitous activator of numerous TATA-containing and TATA-less promoters within the human genome. This Transcription Factor is distinct from several other mammalian activators because it cannot stimulate Transcription of reporter genes when ectopically expressed in Saccharomyces cerevisiae . Here we report that in cultured cells from Drosophila melanogaster human Sp1 efficiently activates Transcription from synthetic promoters containing TATA boxes, but not from promoters that contain an initiator instead of a TATA box. The inability of Sp1 to activate initiator-mediated Transcription did not result from inactivity of the consensus initiator element used for the experiments, as other initiator functions were conserved in Drosophila cells. Interestingly, a difference between the Drosophila and human TFIID complexes was found to be responsible for the selective inability of Sp1 to activate initiator-mediated Transcription in Drosophila; in a complementation assay with a TFIID-depleted HeLa cell extract both the Drosophila and human TFIID complexes supported TATA-mediated Transcription, but only the human complex supported initiator-mediated Transcription. These results suggest that a species-specific interaction is required for activation of TATA-less promoters by Sp1, revealing a difference in Transcriptional activation mechanisms between vertebrates and invertebrates.

  • CIF150, a Human CoFactor for Transcription Factor IID-Dependent Initiator Function
    Molecular and cellular biology, 1998
    Co-Authors: Jörg Kaufmann, Stephen T. Smale, Katharina Ahrens, Ronald Koop, Rolf Müller
    Abstract:

    The Transcription Factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated Factors (TAFs), with the exception of dTAFII150. We previously reported the identification of CIF, an essential coFactor for TFIID-dependent Transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAFII150, and present biochemical evidence that this Factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAFII150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAFII135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

  • Direct recognition of initiator elements by a component of the Transcription Factor IID complex.
    Genes & development, 1994
    Co-Authors: J Kaufmann, Stephen T. Smale
    Abstract:

    A core promoter element called an initiator (Inr) overlaps the Transcription start site of numerous mammalian protein-coding genes. In promoters that lack a TATA box, the Inr is functionally analogous to TATA, in that it is capable of directing basal Transcription by RNA polymerase II and of determining the precise site of Transcription initiation. In promoters that contain a TATA box, the Inr can greatly enhance promoter strength. Mammalian Inr consensus sequences have been defined through functional studies and sequence comparisons of the start site regions of protein-coding genes. Here, we show that, in a DNase I footprinting assay with synthetic promoters, the purified TATA-binding protein complex TFIID specifically contacted the Inr. The TFIID-Inr interaction relies on the precise nucleotides needed for Inr function. Detection of the interaction was dependent either on a TATA box or on Spl bound to upstream sites. Furthermore, recombinant TFIIB appeared to influence the TFIID-Inr interaction, whereas TFIIA stabilized the TFIID-TATA interaction. These results demonstrate that distinct components of TFIID interact with the TATA boxes and lnr elements of core promoters for RNA polymerase II.

Robert G. Roeder - One of the best experts on this subject based on the ideXlab platform.

  • Downstream promoter sequences facilitate the formation of a specific Transcription Factor IID-promoter complex topology required for efficient Transcription from the megalin/low density lipoprotein receptor-related protein 2 promoter.
    The Journal of biological chemistry, 2000
    Co-Authors: Anders Knutson, Robert G. Roeder, Enrique Castaño, Thomas Oelgeschläger, Gunnar Westin
    Abstract:

    Megalin/low density lipoprotein receptor-related protein 2 (LRP-2) is an endocytic receptor expressed in highly specialized cell types such as parathyroid cells and epithelia of the kidney. Previous experiments identified a nonconsensus TATA element, with the sequence TAGAAAA, as crucial for accurate and efficient Transcription from the LRP-2 promoter. Here we show that, in addition to the TAGA element, promoter sequences downstream of the Transcription start site contribute significantly to Transcription both in vitro and in transfected cells. Deletion and point mutational analyses reveal that the promoter region located between positions +5 and +11 (sequence TTTTGGC) is of particular importance. Complementation experiments in nuclear extracts lacking Transcription Factor IID (TFIID) activity show that TATA-binding protein-associated Factors of TFIID are essential for the function of LRP-2 downstream promoter sequences. Interestingly, DNase I footprinting studies show that the downstream region between positions +5 and +11 does not significantly affect overall TFIID affinity to the promoter but that it profoundly affects the topology of the TFIID·promoter complex not only downstream of the Transcription start site, but in particular in the TATA box region. Our observations suggest a model for a novel downstream sequence function, in which TATA-binding protein-associated Factor-promoter interactions downstream of the Transcription start site modulate TFIID-DNA interactions in the TATA box region.

  • Downstream promoter sequences facilitate the formation of a specific Transcription Factor IID-promoter complex topology required for efficient Transcription from the megalin/low density lipoprotein r
    2000
    Co-Authors: Anders Knutson, Robert G. Roeder, Enrique Castaño, Thomas Oelgeschläger, Gunnar Westin
    Abstract:

    Downstream promoter sequences facilitate the formation of a specific Transcription Factor IID-promoter complex topology required for efficient Transcription from the megalin/low density lipoprotein r

  • A human SPT3-TAFII31-GCN5-L acetylase complex distinct from Transcription Factor IID.
    The Journal of biological chemistry, 1998
    Co-Authors: Ernest Martinez, Tapas K. Kundu, Robert G. Roeder
    Abstract:

    Abstract In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with Transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivoeither with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex.

  • Poly(ADP-ribose) polymerase enhances activator-dependent Transcription in vitro
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Michael Meisterernst, Gertraud Stelzer, Robert G. Roeder
    Abstract:

    Mammalian cells contain activities that amplify the effects of activators on class II gene Transcription in vitro. The molecular identity of several of these coFactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive coFactor 1 activity. PARP enhances Transcription by acting during preinitiation complex formation, but at a step after binding of Transcription Factor IID. This Transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding coFactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding coFactors and may point to interactions of PARP with RNA polymerase II-associated Factors in special situations.

  • Cloning and Characterization of Human TAF20/15 MULTIPLE INTERACTIONS SUGGEST A CENTRAL ROLE IN TFIID COMPLEX FORMATION
    The Journal of biological chemistry, 1996
    Co-Authors: Alexander Hoffmann, Robert G. Roeder
    Abstract:

    TFIID is a multiprotein complex that plays a central role in the initiation and regulation of class II Transcription. Transcription Factor IID (TFIID) nucleates Transcription initiation complex formation by direct core promoter binding and mediates the action of Transcriptional activators, in part via direct interactions with them. Molecular studies of the TFIID complex have identified multiple subunits whose potential interactions can be recapitulated in vitro with recombinant polypeptides. Here we report the cloning of human TATA box binding protein (TBP)-associated Factor 20 (TAF20) and the consequent identification of an additional, related TFIID subunit, human TAF15 (hTAF15). Multiple TAF20/15 interactions have been detected within native TFIID preparations and further analyzed with recombinant subunits. Along with the demonstration of a high affinity association between TAF20/15 and TBP, the present results suggest that hTAF20/15 may complement hTAF250 in directing the association of TAFs with TBP to form a TFIID complex. Finally, we present detailed mutagenesis studies that reveal multiple, distinct interaction surfaces on the presumed globular domain of hTAF20/15 and may be used, in conjunction with structural data, to model the architecture of the TFIID multiprotein complex.

Robert Tjian - One of the best experts on this subject based on the ideXlab platform.

  • Core promoter recognition complex changes accompany liver development
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Joseph A. D’alessio, Holger Willenbring, Robert Tjian
    Abstract:

    Recent studies of several key developmental transitions have brought into question the long held view of the basal Transcriptional apparatus as ubiquitous and invariant. In an effort to better understand the role of core promoter recognition and coactivator complex switching in cellular differentiation, we have examined changes in Transcription Factor IID (TFIID) and coFactor required for Sp1 activation/Mediator during mouse liver development. Here we show that the differentiation of fetal liver progenitors to adult hepatocytes involves a wholesale depletion of canonical coFactor required for Sp1 activation/Mediator and TFIID complexes at both the RNA and protein level, and that this alteration likely involves silencing of Transcription Factor promoters as well as protein degradation. It will be intriguing for future studies to determine if a novel and as yet unknown core promoter recognition complex takes the place of TFIID in adult hepatocytes and to uncover the mechanisms that down-regulate TFIID during this critical developmental transition.

  • Three-dimensional structure of the human TFIID-IIA-IIB complex.
    Science (New York N.Y.), 1999
    Co-Authors: Frank Andel, Robert Tjian, Andreas G. Ladurner, Carla Inouye, Eva Nogales
    Abstract:

    The multisubunit Transcription Factor IID (TFIID) is an essential component of the eukaryotic RNA polymerase II machinery that works in concert with TFIIA (IIA) and TFIIB (IIB) to assemble initiation complexes at core eukaryotic promoters. Here the structures of human TFIID and the TFIID-IIA-IIB complex that were obtained by electron microscopy and image analysis to 35 angstrom resolution are presented. TFIID is a trilobed, horseshoe-shaped structure, with TFIIA and TFIIB bound on opposite lobes and flanking a central cavity. Antibody studies locate the TATA-binding protein (TBP) between TFIIA and TFIIB at the top of the cavity that most likely encompasses the TATA DNA binding region of the supramolecular complex.

  • Reconstitution of TATA-binding protein-associated Factor/TATA-binding protein complexes for in vitro Transcription.
    Methods in enzymology, 1996
    Co-Authors: Jin-long Chen, Robert Tjian
    Abstract:

    Publisher Summary This chapter describes strategies for assembling functionally active TAF /TBP (TBP-associated Factors/TATA-binding protein) complexes. The chapter discusses methods developed to build TAF/TBP complexes in vitro . Using this powerful strategy, studies have revealed that different classes of activators function through different mechanisms involving distinct sets of TAFs to regulate Transcription. Moreover, the functional analysis of partial complexes has shown that TAFs are also involved in core promoter recognition in basal Transcription. Reconstitution experiments have suggested that the multisubunit nature of the TFIID (Transcription Factor IID) complex indeed contributes to Transcriptional synergism and core promoter switching, two processes involved in important biological regulatory events. Interestingly, there are additional TBP-containing complexes that consist of distinct TAFs important for Pol I and Pol II Transcription. In addition to TBP-containing complexes, several other Transcription Factors are composed of multiple subunits. For example, TFIIH has been shown to consist of at least 7 polypeptides, while RNA Pol II contains more than 10 subunits.

  • DNA Template and Activator-Coactivator Requirements for Transcriptional Synergism by Drosophila Bicoid
    Science (New York N.Y.), 1995
    Co-Authors: Frank Sauer, Stig K. Hansen, Robert Tjian
    Abstract:

    The template and coactivator requirements for synergistic Transcription directed by a single activator, Bicoid (BCD), bound to multiple sites have been determined. Mutagenesis studies in combination with protein binding experiments and reconstituted Transcription reactions identified two independent activation domains of BCD that target different coactivator subunits (TAF II 110 and TAF II 60) of the basal Transcription Factor IID (TFIID). The presence of both coactivators is required for BCD to recruit the TATA binding protein (TBP)-TAF complex to the promoter and direct synergistic activation of Transcription. Thus, contact between multiple activation domains of BCD and different targets within the TFIID complex can mediate Transcriptional synergism.

  • p53 Transcriptional activation mediated by coactivators TAFII40 and TAFII60
    Science (New York N.Y.), 1995
    Co-Authors: Catherine J. Thut, Jin-long Chen, Richard A. Klemm, Robert Tjian
    Abstract:

    The tumor suppressor protein p53 is a Transcriptional regulator that enhances the expression of proteins that control cellular proliferation. The multisubunit Transcription Factor IID (TFIID) is thought to be a primary target for site-specific activators of Transcription. Here, a direct interaction between the activation domain of p53 and two subunits of the TFIID complex, TAFII40 and TAFII60, is reported. A double point mutation in the activation domain of p53 impaired the ability of this domain to activate Transcription and, simultaneously, its ability to interact with both TAFII40 and TAFII60. Furthermore, a partial TFIID complex containing Drosophila TATA binding protein (dTBP), human TAFII250, dTAFII60, and dTAFII40 supported activation by a Gal4-p53 fusion protein in vitro, whereas TBP or a subcomplex lacking TAFII40 and TAFII60 did not. Together, these results suggest that TAFII40 and TAFII60 are important targets for transmitting activation signals between p53 and the initiation complex.