Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription factor TFIIB is essential for the formation of RNA polymerase II initiation complexes where it binds to the TATA-binding protein (TBP) complex with DNA and recruits RNA polymerase II. TFIIB is probably a target for various activators. Several models have been proposed for the position of TFIIB in the TFIIB-TBP-DNA complex. Here we examine the structure of this complex using gel mobility-shift assays and hydroxyl-radical footprinting. TFIIB requires at least seven base pairs of DNA on either side of the TATA box to form a stable TFIIB-TBP-DNA complex. The sugar residues protected from hydroxyl-radical cleavage by the TFIIB-TBP complex were mapped on the crystal-structure model of the TBP-DNA complex. This analysis suggests that TFIIB binds beneath the concave surface of TBP, contacting DNA both upstream and downstream of the TATA box. Our model predicts that TFIIB binds close to the C-terminal stirrup of TBP and provides one explanation for why TBP needs to bend DNA.
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PMID:Model for binding of transcription factor TFIIB to the TBP-DNA complex. 763 13

RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP.
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PMID:Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. 764 11

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.
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PMID:Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae. 766 72

Levels of mRNA and protein encoded by the TATA-binding protein (tbp) gene are shown to increase dramatically during late spermatogenesis in rodents, culminating in a highly testis-enriched expression pattern. Whereas adult spleen and liver contained roughly 0.7 and 2.3 molecules of TBP mRNA per haploid genome-equivalent, respectively, adult testis contained 80-200 molecules of TBP mRNA per haploid genome-equivalent. Comparison of nuclear and cytoplasmic levels of TBP mRNA in liver and testis suggested that nuclear events (transcription or processing) contribute roughly 12-fold, and cytoplasmic events (mRNA stability) roughly 6-fold, to testis-specific overaccumulation. Levels of nuclear TBP protein in testis cells were, on average, 8- and 11-fold higher than those in liver and spleen cells, respectively. Overexpression of TBP mRNA in testis began about 20 days after birth and reached a plateau around day 40, corresponding to the developmental emergence of haploid cells. Besides TBP, two other components of the general RNA polymerase II machinery, TFIIB and RNA polymerase II, were also overexpressed in testis. By immunostaining, it was found that TBP and RNA polymerase II were particularly rich in round spermatid nuclei. Our results suggest a molecular explanation for how early spermatids are able to accumulate all of the mRNA necessary for the final week of spermiogenesis.
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PMID:High accumulation of components of the RNA polymerase II transcription machinery in rodent spermatids. 767 3

One of the important regulatory concepts to emerge from studies of eukaryotic gene expression is that RNA polymerase II promoters and their upstream activators are composed of functional modules whose synergistic action regulates the transcriptional activity of a nearby gene. Biochemical analysis of synergy by ZEBRA, a non-acidic activator of the Epstein-Barr virus (EBV) lytic cycle, showed that the synergistic transcriptional effect of promoter sites and activation modules correlates with assembly of the TFIID:TFIIA (DA) complex in DNase I footprinting and gel shift assays. The activator-dependent DA complex differs from a basal DA complex by its ability to bind TFIIB stably in an interaction regulated by TATA-binding protein-associated factors (TAFs). TFIIB enhances the degree of synergism by increasing complex stability. Similar findings were made with the acidic activator GAL4-VP16. Our data suggest a unifying mechanism for gene activation and synergy by acidic and non-acidic activators, and indicate that synergy is manifested at the earliest stage of preinitiation complex assembly.
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PMID:A general mechanism for transcriptional synergy by eukaryotic activators. 767 13

We have shown previously that under specific conditions, a TATA box will mediate efficient in vitro transcription by RNA polymerase (pol) III in the absence of a PSE or other promoter elements. The reaction requires a HeLa cell phosphocellulose protein fraction, fraction B, which must be preincubated with the template DNA. Fraction B does not contain any detectable pol II type transcription factor IID (TFIID) activity. In this report, the relationship between fraction B and TFIID was further examined. Purified human TATA-binding protein (TBP) can substitute for fraction B to mediate TATA-dependent pol III transcription. Both TBP and fraction B prefer a reverse TATA box for pol III transcription, yet TBP bound to a reverse TATA box functions poorly for pol II transcription. Like TFIID, fraction B forms a template-committed complex with TATA-containing promoters. TBP, however, will not template commit for pol III transcription unless premixed with phosphocellulose fraction C. TBP-mediated pol III transcription is also more sensitive to the detergent Sarkosyl (N-lauroylsarcosine, Sigma) than is the fraction B reaction unless it is premixed with fraction C. Together, the data suggest that TBP can complex with a component of fraction C, and this complex is then functionally equivalent to fraction B. We propose that fraction B contains TBP in a complex with some other component(s) of the pol III transcription machinery and that this B complex TBP may be specific for pol III transcription.
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PMID:TATA box-mediated in vitro transcription by RNA polymerase III. Evidence for TATA-binding protein in a polymerase III type complex. 767 50

Basal transcription by human RNA polymerase II requires the coordinate action of several ancillary factors (TFIIA-J) and can be regulated by various promoter-specific DNA binding proteins. An additional class of factors, called coactivators, are dispensable for basal transcription but are indispensable for regulation by transcriptional activators. Biochemical studies established that some coactivators are associated with the TATA-binding protein (TBP) to form the TFIID complex. We therefore set out to define the relationship between TBP and these TBP-associated factors (TAFs). Here we describe the cloning, expression and properties of the first human TAF, hTAFII250. The hTAFII250 gene is identical to a gene, CCG1, (ref 7,8), implicated in cell-cycle progression. Recombinant hTAFII250 binds directly to TBP both in vitro and in yeast, and participates in the formation of the TFIID complex. This largest TAF may therefore play a central role in TFIID assembly by interacting with both TBP and other TAFs, as well as serving to link the control of transcription to the cell cycle.
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PMID:Cloning and expression of human TAFII250: a TBP-associated factor implicated in cell-cycle regulation. 768 Jul 71

Two multisubunit complexes containing the TATA-binding protein (TBP) were isolated from HeLa cells constitutively expressing the FLAG epitope-tagged TBP using antibody affinity and peptide elution methods. One of the complexes (f:TFIID), isolated from the P11 0.85 M KCl fraction, contains at least 13 specific TBP-associated factors (TAFs) and can mediate activator-dependent transcription by RNA polymerase II. Importantly, activator function through the highly purified f:TFIID complex still requires a general cofactor fraction containing upstream factor stimulatory activity (USA). As previously observed with partially purified activator-competent natural TFIID, f:TFIID generates extended TATA-dependent footprints on the intrinsically strong adenovirus major late promoter (MLP) but only restricted footprints on the weak adenovirus E1b and E4 and HIV (core) promoters. Along with previous demonstrations of activator-induced downstream TFIID interactions on the E4 promoter, these results argue for a relationship between downstream interactions and overall promoter strength. Initiator-like sequences appear not to be essential for downstream interactions since they have no effect on downstream MLP interactions when mutated, do not effect downstream interactions on the HIV promoter and are not present on the inducible E4 promoter. The other multisubunit complex (f:TFIIIB), isolated from the P11 0.30 M KCl fraction, contains four specific TAFs and can substitute for one of the fractions (TFIIIB) required for RNA polymerase III (pol III) transcription. Neither f:TFIID nor TBP could substitute for this pol III TBP-containing fraction. This plus the fact that f:TFIIIB failed to generate a footprint on the MLP underscores the importance of TAFs in determining promoter specificity by different RNA polymerases.
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PMID:Unique TATA-binding protein-containing complexes and cofactors involved in transcription by RNA polymerases II and III. 768 40

Biochemical analyses have suggested potential targets for transcriptional activation domains, which include several components of the RNA polymerase II machinery, as well as the chromatin template. Here we examine the mechanism of transcriptional activation in yeast cells by connecting a heterologous DNA-binding domain (LexA) to the TATA-binding protein (TBP). LexA-TBP efficiently activates transcription from a promoter containing a LexA operator upstream of a TATA element. Activation is promoter-specific and is sensitive to mutations on the DNA-binding surface of TBP; hence it is not due to a fortuitous activation domain on TBP. Thus a promoter-bound protein lacking an activation domain can stimulate transcription if it is directly connected to TBP. This suggests that recruitment of TBP to the promoter can be a rate-limiting step for transcription in vivo, and that interactions between activation domains and factors that function after TBP recruitment can be bypassed for activation.
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PMID:Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. 772 28

Eukaryotic transcriptional activators may stimulate RNA polymerase II activity by promoting assembly of preinitiation complexes on promoters through their interactions with one or more components of the basal machinery. On the basis of its central role in initiating transcription-complex formation upon binding to the TATA box, the general transcription factor TFIID, which includes the TATA-binding protein (TBP) and several TBP-associated factors, has been implicated as a target for activators. Consistent with this idea, an increasing number of activators have been reported to bind directly to TBP. To assess the functional importance of these in vitro interactions for transcriptional regulation in vivo, we made use of a novel strategy in yeast to show that a physical interaction with TBP is sufficient for a sequence-specific DNA-binding protein to increase initiation of transcription by RNA polymerase II. These results imply that binding of TFIID to promoter elements is a limiting step in transcription complex assembly in vivo.
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PMID:Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo. 772 29


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