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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)

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.
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PMID:In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates. 131 66

We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.
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PMID:A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III. 145 50

We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5'-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed.
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PMID:Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization. 154 3

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.
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PMID:The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II. 156 52

We demonstrate that multiple SP1 protein:DNA binding sites confer enhancer-independent activation on the HIV-1 and globin gene promoters. This activation process can be achieved either by DNA replication of the promoter-containing plasmid or by high concentrations of input plasmid DNA used in the transfections. In the case of HIV-1, the three SP1 sites adjacent to the promoters TATA box are essential for this activation process. Furthermore, the human beta globin gene, which is normally dependent on a linked enhancer for transcriptional activity, can be made enhancer independent by insertion of SP1 binding sites adjacent to its TATA box. We speculate that (SP1)n-TATA type RNA polymerase II promoters may be generally permissive when present on actively replicating DNA templates and that this property of the HIV-1 promoter may be of importance to the activation of the DNA provirus in latently infected T cells.
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PMID:Multiple SP1 binding sites confer enhancer-independent, replication-activated transcription of HIV-1 and globin gene promoters. 162 32

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.
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PMID:Functional analysis of Drosophila transcription factor IIB. 164 95

Consistent with the complexity of the temporally regulated processes that must occur for growth and development of higher eukaryotes, it is now apparent that transcription is regulated by the formation of multicomponent complexes that assemble on the promoters of genes. These complexes can include (in addition to the five or more general transcription factors and RNA polymerase II) DNA-binding proteins, transcriptional activators, coactivators, adaptors and various accessory proteins. The best studied example of a complex that includes a transcriptional adaptor, accessory proteins and a DNA-binding protein is that involving the herpes simplex virus VP16 protein. Evidence suggests that the adenovirus E1a protein and the cellular Sp1 and CTF/NF1 transcription factors also function through adaptors or coactivators. Each additional component of the transcription complex provides the cell with another point at which to exert control of gene expression.
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PMID:The interactions of transcription factors and their adaptors, coactivators and accessory proteins. 166 80

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.
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PMID:Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. 171 39

Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells. We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site. Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species. The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication. Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP-response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences. The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box. The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA.
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PMID:Nucleotide sequence of murine PCNA: interspecies comparison of the cDNA and the 5' flanking region of the gene. 172 65

We have identified a component of the eukaryotic RNA polymerase II transcriptional machinery that is more heat-labile than TFIID. DHFR transcriptional activity was severely reduced in 40 degrees C heat-treated extracts in which TFIID was fully active. This heat-labile activity was required for the transcription of both TATA box and non-TATA box promoters that are activated by the transcription factor Sp1. Gel mobility shifts indicated that Sp1 DNA binding activity was heat-labile, and the addition of purified Sp1 to 40 degrees C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Sp1 to 47 degrees C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Sp1 DNA binding activity is partially responsible for the heat-sensitive loss of DHFR transcriptional activity, but that a second essential activity is also inactivated by 47 degrees C heat-treatment. The discovery of this heat-labile component of Sp1 activation has two important implications in the analysis of transcriptional regulation. First, it demonstrates that heat-treated extracts are not appropriate for examination of the involvement of TFIID in the transcription of Sp1-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Sp1-activated promoters should not be performed at 30 degrees C.
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PMID:Sp1 activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component. 182 Feb 11


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