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Query: UNIPROT:P20226 (TATA-binding protein)
 1,297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.
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PMID:TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4. 968 14

Purification of multiprotein complexes such as transcription factor (TF) IIH and RNA polymerase II (pol II) has been a tedious task by conventional chromatography. To facilitate the purification, we have developed an effective scheme that allows human TFIIH and pol II to be isolated from HeLa-derived cell lines that conditionally express the FLAG-tagged p62 subunit of human TFIIH and the RPB9 subunit of human pol II, respectively. An approximate 2000-fold enrichment of FLAG-tagged TFIIH and a 1000-fold enhancement of total pol II are achieved by a one-step immunoaffinity purification. The purified complexes are functional in mediating basal and activated transcription, regardless of whether TATA-binding protein or TFIID is used as the TATA-binding factor. Interestingly, repression of basal transcription by the positive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH, and pol II holoenzyme, suggesting that phosphorylation of PC4 by these proteins may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, pol II complexes with different phosphorylation states on the carboxyl-terminal domain of the largest subunit are selectively purified from the inducible pol II cell line, making it possible to dissect the role of carboxyl-terminal domain phosphorylation in the transcription process in a highly defined in vitro transcription system.
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PMID:Immunoaffinity purification and functional characterization of human transcription factor IIH and RNA polymerase II from clonal cell lines that conditionally express epitope-tagged subunits of the multiprotein complexes. 985 12

The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter.
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PMID:Activation of the murine dihydrofolate reductase promoter by E2F1. A requirement for CBP recruitment. 1033 93

A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription.
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PMID:DNA bending and wrapping around RNA polymerase: a "revolutionary" model describing transcriptional mechanisms. 1035 58

The Rad25 protein in yeast is a DNA helicase and a subunit of the general transcription factor TFIIH. While in vitro studies have led to the hypothesis that TFIIH helicase activity plays a role in promoter melting, in vivo tests are lacking. Using potassium permanganate, which preferentially modifies single-stranded DNA, we show that a temperature-sensitive rad25(ts) mutant severely reduces the normally extensive promoter melting observed in vivo on the highly expressed genes TDH2 and PDC1 and on the induced heat shock gene HSP82. Loss of promoter melting can be observed in as little as 30 s after a shift to the nonpermissive temperature and is accompanied by a dramatic reduction in transcription. These effects on the promoter are specific, since the mutation does not affect TATA box occupancy or, in the case of HSP82, recruitment of TATA-binding protein to the TATA element or that of heat shock factor to heat shock elements. Additionally, using the technique of formaldehyde cross-linking coupled with restriction endonuclease cleavage and ligation-mediated PCR, we were able to map the polymerase density on the promoter of HSP82. This high-resolution mapping allowed us to determine that the polymerase II (Pol II) density on the promoter is also dramatically reduced after inactivation of TFIIH. These data provide strong support for the hypothesis that TFIIH functions with Pol II in the transcriptionally required step of promoter melting and show, surprisingly, that the extent of TFIIH-dependent promoter melting observed in vivo is several times larger than that seen in vitro.
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PMID:Transcription factor TFIIH is required for promoter melting in vivo. 1040 54

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.
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PMID:TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism. 1056 21

The yeast Gal11 protein is an important component of the Mediator complex in RNA polymerase II-directed transcription. Gal11 and the general transcription factor (TF) IIE are involved in regulation of the protein kinase activity of TFIIH that phosphorylates the carboxyl-terminal domain of RNA polymerase II. We have previously shown that Gal11 binds the small and large subunits of TFIIE at two Gal11 domains, A and B, respectively, which are important for normal function of Gal11 in vivo. Here we demonstrate that Gal11 binds directly to TFIIH through domain A in vitro. A null mutation in GAL11 caused lethality of cells when combined with temperature-sensitive mutations in the genes encoding TFIIE or the carboxyl-terminal domain kinase, indicating the presence of genetic interactions between Gal11 and these proteins. Mutational depletion of Gal11 or TFIIE caused inefficient opening of the transcription initiation region, but had no significant effect on TATA-binding protein occupancy of the TATA sequence in vivo. These results suggest that the functions of Gal11 and TFIIE are necessary after recruitment of TATA-binding protein to the TATA box presumably at the step of stable preinitiation complex formation and/or promoter melting. We illustrate genetic interactions between Gal11 and other Mediator components such as Med2 and Pgd1/Hrs1/Med3.
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PMID:Functional connections between mediator components and general transcription factors of Saccharomyces cerevisiae. 1097 56

Transcription factor (TF) IID, comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), is a general transcription factor required for RNA polymerase II (pol II) transcription on most eukaryotic genes. Recent findings that TAFs may not be globally required for activator-dependent transcription in vivo and in vitro and that both TAF-dependent and TAF-independent promoters are found in yeast suggest that transcriptional activation can occur through at least two different pathways, depending on the presence or absence of TAFs. Using order-of-addition and template challenge assays performed in a human cell-free transcription system reconstituted with recombinant general transcription factors (TFIIB, TBP, TFIIE, TFIIF), a recombinant general cofactor (PC4), and highly purified epitope-tagged multiprotein complexes (TFIID, TFIIH, pol II), we demonstrate that when TBP is used as the TATA-binding factor transcriptional activators such as Gal4-VP16 and human papillomavirus E2 mainly function by facilitating pol II entry to the promoter region. In contrast, when TFIID is used as the TATA-binding factor, promoter recognition by TFIID appears to be the rate-limiting step facilitated by transcriptional activators during preinitiation complex assembly. Using protein-protein pull-down and far-Western analyses, we further show that the presence of TAFs in TFIID facilitates the recruitment of pol II by transcriptional activators, thereby switching the rate-limiting step from pol II entry to promoter recognition. Our findings thus provide distinct molecular mechanisms for TAF-independent and TAF-dependent activation.
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PMID:TATA-binding protein-associated factors enhance the recruitment of RNA polymerase II by transcriptional activators. 1145 28

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.
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PMID:Mediator influences Schizosaccharomyces pombe RNA polymerase II-dependent transcription in vitro. 1453 14

The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors. In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II. Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3' portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions. Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters. Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized. We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.
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PMID:The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo. 1458 89


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