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D Reinberg

Publications and source records attributed to D Reinberg.

At least 73 records · Page 4Linked to original sources
PubMed1995

Solution structure of the C-terminal core domain of human TFIIB: similarity to cyclin A and interaction with TATA-binding protein.

TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIBc) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar alpha-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic alpha helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIBc and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries.

Amino Acid Sequence↗
PubMed1995

Recycling of the general transcription factors during RNA polymerase II transcription.

We have analyzed the fate of the RNA polymerase II (RNAPII) general transcription factors during the transition from initiation to elongation using multiple approaches. We demonstrate that all of the basal factors coexist in mature initiation complexes but that following nucleotide addition, this complex becomes disrupted. During this transition, TFIID remains promoter-bound whereas TFIIB, TFIIE, TFIIF, and TFIIH are released. Upon release, TFIIB reassociates with TFIID, reforming the RNAPII docking site, the DB complex. TFIIE is released before formation of the tenth phosphodiester bond. This precedes TFIIH release, which occurrs after the transcription complex reaches +30. TFIIF is unique in that it is the only basal factor detected in the RNAPII elongation complex. Following its release from the initiation complex, TFIIF has the ability to reassociate with a stalled RNAPII.

Adenoviridae↗
PubMed1995

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

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.

Adenosine Triphosphatases↗
PubMed1995

Cdk-activating kinase complex is a component of human transcription factor TFIIH.

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). 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.

Amino Acid Sequence↗
PubMed1995

Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters.

The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples. Excellent agreement among the three methods was obtained. With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed. Non-specific binding to DNA ends was common. The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization. With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end. Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees. Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions.

Adenoviridae↗
PubMed1995

Transcriptional activation. Tuning-up transcription.

Eukaryotic transcription is a complex process, and recent results identify multiple steps that need to be stimulated to activate transcription, one of which is a change in conformation of the general transcription factor TFIIB.

Animals↗
PubMed1995

News on initiation and elongation of transcription by RNA polymerase II.

Transcription by RNA polymerase II is a complex process that requires additional factors to initiate transcription at the promoters. New developments in the past year have furthered our understanding of the functions of the transcription factors and provided more insights into the mechanisms involved in the regulation of initiation and elongation of transcription. One of the most significant advances of the past year was the discovery of the involvement of the general transcription factor TFIIH in DNA excision repair. Surprisingly, studies aimed at identifying the kinase activity within TFIIH responsible for phosphorylating the carboxy-terminal domain of RNA polymerase II revealed it to be the MO15/Cdk7 kinase and its partner, cyclin H. These exciting observations suggest a paradigm for linking transcription, DNA excision repair and cell cycle progression through one pivotal factor.

Humans↗
PubMed1995

Common themes in assembly and function of eukaryotic transcription complexes.

Eukaryotes contain three distinct RNA polymerase enzymes, each responsible for the transcription of a subclass of nuclear genes. Despite this division of labor, each RNA polymerase system follows a common blueprint to execute the loading of the polymerase onto the relevant promoter region. The RNA polymerase II system appears unique in that after RNA polymerase II has loaded onto the DNA, two auxiliary factors, TFIIE and TFIIH, are necessary for its escape from the promoter region. The complexity of the RNA polymerase II initiation pathway provides a multitude of potential targets for transcriptional activators. Tight control over transcription initiation levels is afforded by multiple cofactors that both enhance and repress.

Animals↗
PubMed1994

Differential regulation of RNA polymerases I, II, and III by the TBP-binding repressor Dr1.

RNA polymerases I, II, and III each use the TATA-binding protein (TBP). Regulators that target this shared factor may therefore provide a means to coordinate the activities of the three nuclear RNA polymerases. The repressor Dr1 binds to TBP and blocks the interaction of TBP with polymerase II- and polymerase III-specific factors. This enables Dr1 to coordinately regulate transcription by RNA polymerases II and III. Under the same conditions, Dr1 does not inhibit polymerase I transcription. By selectively repressing polymerases II and III, Dr1 may shift the physiological balance of transcriptional output in favor of polymerase I.

Base Sequence↗
PubMed1994

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.

Amino Acid Sequence↗
PubMed1994

Structure-function analysis of the TBP-binding protein Dr1 reveals a mechanism for repression of class II gene transcription.

Dr1, a repressor of class II genes, regulates transcription by a novel mechanism. Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex. By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo. In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays. Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo. By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression. One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues. The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression. We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo.

Amino Acid Sequence↗
PubMed1994

Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.

Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex.

Adenovirus E1A Proteins↗
PubMed1994

Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II.

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.

DNA Helicases↗
PubMed1994

The multifunctional TFIIH complex and transcriptional control.

RNA polymerase II (Pol II) requires seven general transcription factors (GTFs) and ATP for transcription initiation. Transcription factor IIH (TFIIH) has emerged as the sole GTF with enzymatic activity. In addition to its essential role in transcription initiation, recent studies have demonstrated a direct involvement of TFIIH in DNA excision repair processes. The enzymatic properties and functional duality of TFIIH make it a prime target for regulation by viral and cellular factors.

Amino Acid Sequence↗
PubMed1994

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

Fungal Proteins↗
PubMed1993

Interaction of human thyroid hormone receptor beta with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone.

The human thyroid hormone receptor beta (hTR beta) is capable of both transcriptional silencing and hormone-dependent activation. However, the detailed mechanism of this transcriptional regulation remains to be elucidated. One possibility is that hTR beta interacts directly with factors of the basal transcriptional machinery, thereby modulating basal promoter activity in a direct manner, as has been shown for other transcription factors. Here, we show that hTR beta interacts specifically with the human basal transcription factor TFIIB. Deletion analysis revealed two contact sites in the receptor: one is located in the N terminus, while the other is part of the ligand-binding domain (LBD) and is located at the C terminus. Interestingly, each receptor contact site interacts with different sites in TFIIB. Cotransfection experiments revealed that, when fused to the DNA-binding domain of yeast transcription factor GAL4, the C-terminal interaction site of hTR beta was transcriptionally inactive; however, when it was cotransfected with the remaining part of the LBD on a separate molecule, silencing function was restored. In agreement with that, we show that thyroid hormone is able to significantly decrease the interaction of its receptor LBD with TFIIB. Our data suggest that hTR beta acts as a transcriptional silencer by interacting with TFIIB and that thyroid hormone may act in part by preventing transcriptional repression at this level.

Actins↗