Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P20226 (TATA-binding protein)
 1,297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human T-lymphotropic virus type I (HTLV-I) promoter contains the structural features of a typical RNA polymerase II (pol II) template. The promoter contains a TATA box 30 bp upstream of the transcription initiation site and binding sites for several pol II transcription factors, and long poly(A)+ RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I transcription activity was reconstituted in vitro by using TATA-binding protein, TFIIA, recombinant TFIIB, TFIIE, and TFIIF, TFIIH, and pol II. Transcription of the HTLV-I promoter in the reconstituted system requires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I long terminal repeat also contains an overlapping transcription unit (OTU). HTLV-I OTU transcription is initiated at the same nucleotide site as the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of alpha-amanitin at concentrations which inhibited the adenovirus major late pol II promoter (6 micrograms/ml). HTLV-I transcription was inhibited when higher concentrations of alpha-amanitin (60 micrograms/ml) were used, in the range of a typical pol III promoter (VA-I). Neutralization and depletion experiments with three distinct pol II antibodies demonstrate that RNA pol II is not required for HTLV-I OTU transcription. Antibodies to basal transcription factors TATA-binding protein and TFIIB, but not TFIIIC, inhibited HTLV-I OTU transcription. These observations suggest that the HTLV-I long terminal repeat contains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors.
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PMID:Transcription of the human T-cell lymphotropic virus type I promoter by an alpha-amanitin-resistant polymerase. 752 15

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 Tax1 gene product has been shown to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax1 transactivation, we have developed an in vitro transactivation assay in which wild-type Tax1 is able to specifically transactivate a polymerase II promoter through upstream Tax1-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax1 specifically transactivates this template 5- to 10-fold in a concentration-dependent manner. No transactivation of the ovalbumin promoter (pLovTATA) template control was observed. Tax1 transactivation was inhibited by low concentrations of alpha-amanitin and was effectively neutralized by anti-Tax1 but not control sera. Consistent with in vivo transactivating activity, Tax1 NF-kappa B mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp repeat. In a reconstituted in vitro transcription assay, Tax1 transactivation was dependent upon basal transcription factors TFIIB, TFIIF, Pol II, TFIID, and TFIIA. TATA-binding protein did not functionally substitute for TFIID in the transactivation assay by Tax1 but was sufficient for basal transcription. Finally, we have used anti-TFIIA antibody (p55) to ask if Tax1 transactivation required TFIIA activity. Addition of TFIIA antibody to in vitro transcription reactions, as well as depletion of TFIIA by preclearing with antibody, showed that TFIIA was required for Tax1 transactivation. Only a slight (twofold) drop of basal transcription was observed. Tax1 transactivation was restored when purified HeLa TFIIA was added back into the reconstituted system. We propose that Tax1 utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.
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PMID:Transactivation of the human T-cell lymphotropic virus type 1 Tax1-responsive 21-base-pair repeats requires Holo-TFIID and TFIIA. 760 77

The minimal requirements for transcription initiation from supercoiled templates were determined for the two major forms of TATA-binding factors found in cell extracts, the 300-kDa B-TFIID and the 1000-kDa D-TFIID complexes. As had been observed for the TATA-binding protein (TBP) subunit (Parvin and Sharp, 1993), transcription from the IgH promoter minimally requires TFIID activity plus TFIIB and RNA polymerase II. This minimal reaction is only active on negatively supercoiled template DNA. In contrast, the supercoiled templates encoding the adenovirus major late promoter (MLP), or several other promoters, require the addition of TFIIF to the minimal reaction. Further addition of TFIIE and TFIIH boosts the level of transcription from these latter promoters but is not required. In contrast to the complete reaction on linear template, transcription from supercoiled IgH or MLP templates does not require the hydrolysis of the beta-gamma bond of ATP. Fourteen different core promoters were compared in complete and minimal basal transcription reactions reconstituted with one of the three TATA activities: TBP, B-TFIID, and D-TFIID. Of these 14 promoters, only the IgH was active in the absence of TFIIF, and the other promoters demonstrated different levels of transcription depending on which basal factors were present in reaction. It is proposed that a significant level of basal transcription only requires a minimal set of factors, and stimulation by upstream activators may in part be mediated by the inclusion of additional basal factors into the initiation reaction.
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PMID:Multiple sets of basal factors initiate transcription by RNA polymerase II. 803 89

Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.
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PMID:Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. 815 90

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.
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PMID:DNA topology and a minimal set of basal factors for transcription by RNA polymerase II. 849 Sep 64

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in TFIIB. Using the gel mobility-shift assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic alpha-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic alpha-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger.
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PMID:Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II. 850 27

Transcription factor TFIIB is an essential component of the RNA polymerase II initiation complex. TFIIB carries out at least two functions: it interacts directly with the TATA-binding protein (TBP) and helps to recruit RNA polymerase II into the initiation complex. The sequence of TFIIB reveals a potential zinc-binding domain and an imperfect duplication of approximately 70 amino acids. Mutagenesis of cysteine codons within the putative zinc finger results in mutant proteins that bind normally to TBP but are unable to recruit RNA polymerase II-TFIIF into the initiation complex. Changing the two most highly conserved amino acids in the TFIIB repeats reduces the ability of TFIIB to interact with TBP. Therefore, the two functions of TFIIB can be assigned to two separable functional domains of the protein.
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PMID:Functional domains of transcription factor TFIIB. 851 12

ICP4 of herpes simplex virus is responsible for the activation of viral transcription during infection. It also efficiently activates and represses transcription in vitro depending on the promoter context. The contacts made between ICP4 and the cellular proteins that result in activated transcription have not been identified. The inability of ICP4 to activate transcription with TATA-binding protein in place of TFIID and the requirement for an initiator element for efficient ICP-4-activated transcription suggest that coactivators, such as TBP-associated factors, are involved (B. Gu and N. DeLuca, J. Virol. 68:7953-7965, 1994). In this study we showed that ICP4 activates transcription in vitro using an immunopurified TFIID, indicating that TBP-associated factors may be a sufficient subset of coactivators for ICP4-activated transcription. Similar to results seen in vivo, the presence of the ICP4 C-terminal region (amino acids 774 to 1298) was important for activation in vitro. With epitope-tagged ICP4 molecules in immunoaffinity experiments, it was shown that the C-terminal region was also required for ICP4 to interact with TFIID present in a crude transcription factor fraction. In the same assay, ICP4 was unable to interact with the basal transcription factors, TFIIB, TFIIE, TFIIF, and TFIIH and RNA polymerase II. ICP4 could also interact with TBP, independent of the C-terminal region. However, reflective of the interaction between ICP4 and TFIID, the ICP4 C-terminal region was required for an interaction with FAF250-TBP complexes and with TAF250 alone. Therefore, the interfaces or conformation of TBP mediating the interaction between ICP4 and TBP in solution is probably masked when TBP is bound to TAF250. With a series of mutant ICP4 molecules purified from herpes simplex virus-infected cells, it was shown that ICP4 molecules that can bind DNA and interact with TAF250 could activate transcription. Taken together, these results demonstrate that ICP4 interaction with TFIID involves the TAF250 molecule and the C-terminal region of ICP4 and that this interaction is part of the mechanism by which ICP4 activates transcription.
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PMID:Interaction of the viral activator protein ICP4 with TFIID through TAF250. 864 20

The aryl hydrocarbon (or dioxin) receptor (AhR) is a ligand-activated basic helix-loop-helix (bHLH) protein that heterodimerizes with the bHLH protein AhR nuclear translocator (ARNT) to form a complex that binds to xenobiotic regulatory elements in the enhancers of target genes. We used a series of fusion proteins, with a heterologous DNA-binding domain, to study independently the trans-activating function of the human AhR and ARNT proteins in yeast. The results confirm that both the human AhR and ARNT contain carboxyl-terminal trans-activation domains. The AhR has a complex trans-activation domain that is composed of multiple segments that function independently and exhibit varying levels of activation. Furthermore, these regions within the AhR cooperate when linked together, resulting in a synergistic activation of transcription. Fusion proteins of the AhR and ARNT trans-activation domains with the LexA DNA-binding domain, expressed in bacteria and purified to near-homogeneity, stimulated transcription of a minimal promoter in vitro in yeast nuclear extracts. Using this in vitro transcription assay, it was also possible to demonstrate that the AhR and ARNT trans-activation domains, in the absence of a DNA-binding domain, inhibited activated and basal transcription. Furthermore, in vitro the receptor bound selectively to the basal transcription factors, the TATA-binding protein and TFIIF, whereas ARNT bound preferentially to TFIIF. Taken together, these results suggest that AhR and ARNT activate target gene expression, at least in part, through direct interactions with basal transcription factors.
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PMID:Trans-activation by the human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins: direct interactions with basal transcription factors. 879 92

In yeast cells, mutations in the TATA-binding protein (TBP) that disrupt the interaction with the TATA element or with TFIIA can selectively impair the response to acidic activator proteins. We analyzed the transcriptional properties of TBP derivatives in which residues that directly interact with TFIIB were replaced by alanines. Surprisingly, a derivative with a 50-fold defect in TBP-TFIIB-TATA complex formation in vitro (E188A) supports viability and responds efficiently to activators in vivo. The E186A derivative, which displays a 100-fold defect in TBP-TFIIB-TATA complex formation, does not support viability, yet it does respond to activators. Conversely, the L189A mutation, which has the mildest effect on the interaction with TFIIB (10-fold), can abolish transcriptional activation and cell viability when combined with mutations on the DNA-binding surface. This "synthetic lethal" effect is not observed with E188A, suggesting that the previously described role of L189 in transcriptional activation may be related to its location on the DNA-binding surface and not to its interaction with TFIIB. Finally, when using TBP mutants defective on multiple interaction surfaces, we observed synthetic lethal effects between mutations on the TFIIA and TFIIB interfaces but found that mutations implicated in association with polymerase II and TFIIF did not have significant effects in vivo. Taken together, these results argue that, unlike the TBP-TATA and TBP-TFIIA interactions, the TBP-TFIIB interaction is not generally limiting for transcriptional activation in vivo.
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PMID:A severely defective TATA-binding protein-TFIIB interaction does not preclude transcriptional activation in vivo. 903 60


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