Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation. Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro. Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16. Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex. Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.
 ...
PMID:Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. 131 Mar 61

The ability of the c-Jun protein, the main component of the transcription factor AP1, to interact directly or indirectly with the RNA polymerase II-initiation complex to activate transcription was investigated by in vivo transcription interference ("squelching") experiments. Coexpression of a Jun mutant lacking its DNA binding domain strongly represses the activity of wild-type c-Jun. Repression depends on the presence of the transactivation domains (TADs), suggesting that a limiting factor interacting with the TADs is essential to link Jun and the components of the transcriptional machinery. The activity of this intermediary factor(s) is restricted to TADs characterized by an abundance of negatively charged amino acids, as demonstrated by the abilities of the TADs of JunB, GAL4, and VP16 to repress c-Jun activity. Depending on the presence of the TADs of Jun, we found physical interaction between Jun and a cluster of three proteins with molecular masses of 52, 53, and 54 kDa (p52/54). Association between Jun and p52/54 is strongly reduced in the presence of VP16, suggesting that the two proteins compete for binding to p52/54. Transcription factors containing a different type of TAD (e.g., GHF1, estrogen receptor, or serum response factor) fail to inhibit Jun activity, suggesting that these proteins act through a different mechanism. We consider the requirement of Jun to interact with p52/54 utilized by other transcription factors a new mechanism in the regulation of transcription of Jun-dependent target genes.
 ...
PMID:A common intermediary factor (p52/54) recognizing "acidic blob"-type domains is required for transcriptional activation by the Jun proteins. 144 82

The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting. The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step. This directs the templates into a productive assembly pathway. Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways. These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis.
 ...
PMID:The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA. 151 30

A Drosophila cDNA encoding a human transcription factor TFIIB homologue was isolated by PCR methods. The deduced amino acid sequence indicates 85% sequence similarity with human TFIIB, and the corresponding cDNA product expressed in Escherichia coli is interchangeable with human TFIIB for both basal and GAL4-VP16-induced transcription. Structural motifs including the direct repeats, basic repeats, and sigma sequence similarities are well conserved among Drosophila, human, and Xenopus TFIIB. However, the N-terminal region of each direct repeat is less conserved among the three species, suggesting the presence of two structural subdomains in the direct repeat. Moreover, the amino acid changes in the N-terminal subdomain produce altered positions of the conserved amino acids between the direct repeats. An overall similarity in general structural features between TFIIB and TFIID tau (the TATA-binding subunit of TFIID) was previously noted. However, in contrast to the sequence divergence reported for the N-terminal domains of TFIID tau from different species, the N-terminal sequence of TFIIB was highly conserved among the species. This suggests that TFIIB has a more rigid structure, consistent with its function as a "bridging" protein between TFIID and RNA polymerase II. Further implications of the TFIIB structure are discussed.
 ...
PMID:Isolation and characterization of a cDNA encoding Drosophila transcription factor TFIIB. 155 90

We have isolated a cDNA encoding Drosophila transcription factor IIB (dTFIIB) and characterized the properties of recombinant dTFIIB with a reconstituted in vitro transcription system derived from Drosophila embryos. Purified, recombinant dTFIIB is fully active at a concentration of one molecule per template DNA. With different promoters, the transcriptional activity of dTFIIB was similar but not identical to that of human TFIIB, which suggests that there may be variations in the mechanisms by which TFIIB functions in transcription. We have also found that recombinant dTFIIB suppressed nonspecific initiation of transcription by RNA polymerase II by a mechanism that appears to involve direct interaction between TFIIB and the polymerase. Addition of excess dTFIIB to transcription reactions resulted in promoter-specific repression of transcription. These experiments have led to the hypothesis that TFIIB interacts with a basal transcription factor that is required for transcription of some, but not all, genes and that the presence of excess dTFIIB results in sequestration of the promoter-specific basal factor to prevent its assembly into a productive transcription complex. Excess dTFIIB did not, however, affect the ability of either GAL4-VP16 or Sp1 to stimulate transcription. These data indicate that in contrast to current models, GAL4 derivatives do not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex.
 ...
PMID:Functional analysis of Drosophila transcription factor IIB. 164 95

The relation between chromatin structure and transcriptional activity was examined by in vitro transcription analysis of chromatin reconstituted in the absence or presence of histone H1. To maintain well-defined template DNA, purified components were used in the reconstitution of chromatin. Reconstitution of nucleosomal cores to an average density of 1 nucleosome per 200 base pairs of DNA resulted in a mild reduction of basal RNA polymerase II transcription to 25 to 50 percent of that obtained with naked DNA templates. This nucleosome-mediated repression was due to nucleosomal cores located at the RNA start site and could not be counteracted by the sequence-specific transcription activators Sp1 and GAL4-VP16. When H1 was incorporated into the chromatin at 0.5 to 1.0 molecule per nucleosome (200 base pairs of DNA), RNA synthesis was reduced to 1 to 4 percent of that observed with chromatin containing only nucleosomal cores, and this H1-mediated repression could be counteracted by the addition of Sp1 or GAL4-VP16 (antirepression). With naked DNA templates, transcription was increased by a factor of 3 and 8 by Sp1 and GAL4-VP-16, respectively (true activation). With H1-repressed chromatin templates, however, the magnitude of transcriptional activation mediated by Sp1 and GAL4-VP16 was 90 and more than 200 times higher, respectively, because of the combined effects of true activation and antirepression. The data provide direct biochemical evidence that support and clarify previously proposed models in which there is depletion or reconfiguration of nucleosomal cores and histone H1 at the promoter regions of active genes.
 ...
PMID:Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. 171 39

The U2 snRNA genes, which are transcribed by RNA polymerase II at high levels in all tissues examined, require both a distal and a proximal sequence element for efficient expression. The distal sequence element which has many properties in common with transcriptional enhancers contains, in addition to Sp1 binding sites, an octamer binding site which mediates activation through interactions with the ubiquitous transcription factor Oct-1. In the present study we have attempted to answer the question whether Oct-1 contains a unique activating domain which is required for activation of snRNA genes or whether ubiquitously expressed and lymphoid specific octamer binding factors both have the capacity to activate snRNA transcription. Our results show that in the presence of Oct-1, overexpression of Oct-2A in HeLa or COS1 cells neither inhibits nor stimulates transcription of U2 constructions which contain octamer binding sites with or without an adjacent Sp1 binding site. Moreover, an Oct-2A--GAL4 fusion protein in which the DNA binding domain of Oct-2A was substituted for by the one of the yeast transcription activator GAL4 activates transcription of a human U2 snRNA gene in which the octamer binding site was replaced by a GAL4 binding site. From the results it is concluded that both Oct-1 and Oct-2A contain domains which can activate the ubiquitously expressed U2 snRNA genes.
 ...
PMID:Both Oct-1 and Oct-2A contain domains which can activate the ubiquitously expressed U2 snRNA genes. 182 77

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.
 ...
PMID:Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae. 187 38

To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.
 ...
PMID:Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. 189 87

In prokaryotes and eukaryotes many gene activators work synergistically. For example, two dimers of lambda repressor interact to promote binding of these proteins to DNA, a reaction that is crucial at the repressor concentrations found in lysogens. In this case one of the bound dimers activates transcription, evidently by touching RNA polymerase. In another example, the yeast transcriptional activator GAL4, which can stimulate transcription in many eukaryotes, binds to multiple sites on DNA to activate transcription synergistically; the presence of two such sites can elicit a level of transcription more than twice that found with a single site. In this paper we show that synergistic activation by each of several GAL4 derivatives involves a mechanism different from that illustrated by the lambda repressor: multiple activator molecules can work synergistically under conditions in which their binding sites on DNA are saturated. The accompanying paper shows that under similar conditions of activator excess, GAL4 derivatives work synergistically with a heterologous mammalian gene activator. These results support the idea that eukaryotic activators can cooperate not by directly interacting but by simultaneously touching some component(s) of the transcriptional machinery.
 ...
PMID:A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. 216 Jun 9


1 2 3 4 5 6 7 8 9 10 Next >>