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)

The TATA box-binding factor TFIID is an essential component for the initiation of transcription by eukaryotic RNA polymerase II. We investigated the effect of DNA supercoiling on TFIID: promoter interactions using recombinant yeast (ry) TFIID. DNase I footprinting analysis showed that ryTFIID has a higher affinity for the adenovirus major late promoter in the negatively supercoiled state than that in the relaxed state. On the contrary, its affinity for the Drosophila hsp70 promoter is constant irrespective of DNA topology. Binding of ryTFIID to these promoters induced underwinding of duplex DNA. The functional TATA box and active ryTFIID are essential for the underwinding. The step was facilitated by negative supercoiling of DNA on the adenovirus major late promoter but not on the Drosophila hsp70 promoter.
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PMID:Underwinding of DNA on binding of yeast TFIID to the TATA element. 850 7

To study of structure of RNA polymerase (pol) II transcription units a nd the influence of temperature on the regulation of gene expression in Trypanosoma brucei, and hsp70 intergenic region promoter was characterized. In T. brucei, the hsp70 locus contains, from 5' to 3', a cognate hsp70-related gene (gene 1) which is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2 to 6). Transcription proceeds on the entire 23-kb locus, and polycistronic transcription occurs in hsp70 genes 2 to 6. Transcription of hsp70 genes 2 to 6 is only moderately sensitive to UV irradiation, indicating that it cannot be driven by a single far-upstream promoter, which suggests that promoters could be located in the region close to the hsp70 coding region. Transient transformations demonstrated that sequences located upstream of hsp70 gene 2 and in the intergenic region between hsp70 genes 2 and 3 are able to direct transcription of the reporter gene, the chloramphenicol acetyltransferase (CAT) gene. The plasmid DNA driven by the hsp70 intergenic region promoter gave CAT activity approximately 85-fold above to background level. This is equivalent to approximately 1% of that derived from a CAT plasmid driven by the procyclic acidic repetitive protein gene promoter, which is controlled by RNA pol I. The hsp70 intergenic region promoter can drive alpha-amanitin-sensitive transcription at an internal position of the chromosome as well as an episome, suggesting that it is controlled by RNA pol II. However, this hsp70 intergenic region promoter, along with the 3' splice site and the 5' untranslated region of the hsp70 genes that controls the transcription of the reporter gene, cannot up-regulate the expression of the reporter gene during heat shock. This result is consistent with the previous observation that expression of the hsp70 genes in T. brucei is mainly controlled at the posttranscriptional level.
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PMID:An RNA polymerase II promoter in the hsp70 locus of Trypanosoma brucei. 862 66

RNA polymerase was purified from vegetative-phase mycelia of Streptomyces griseus by a series of ion-exchange chromatographies. By western blot analysis using antiserum against S. coelicolor HrdB, which is a principal sigma factor (sigma(hrdB)), the purified holoenzyme was found to contain sigmaB (=sigma(hrdB)) of S. griseus. Significant amounts of HrdB protein were, however, eluted from the DEAE column at lower concentrations of KCl than that required for for elution of the holoenzyme containing sigmaB, suggesting that sigmaB is dissociated from the core enzyme, or an excess amount of sigmaB exists in S.griseus cells. The holoenzyme containing sigmaB (EsigmaB) transcribed in vitro the dagA promoter of S. coelicolor, and the hardB and hsp70 promoters of S. griseus, suggesting that it is involved in transcription of the essential genes. EsigmaB may be a major form of RNA polymerase holoenzyme in the growing phase of S. griseus.
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PMID:Purification and characterization of RNA polymerase holoenzyme (E sigma B) from vegetative-phase mycelia of Streptomyces griseus. 869 Jul 6

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.
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PMID:Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract. 881 56

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation.
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PMID:Disruption of downstream chromatin directed by a transcriptional activator. 938 44

Drosophila heat shock factor (HSF) binds to specific sequence elements of heat shock genes and can activate their transcription 200-fold. Though HSF has an acidic activation domain, the mechanistic details of heat shock gene activation remain undefined. Here we report that HSF interacts directly with the general transcription factor TBP (TATA-box binding protein), and these two factors bind cooperatively to heat shock promoters. A third factor that binds heat shock promoters, GAGA factor, also interacts with HSF and further stabilizes HSF binding to heat shock elements (HSEs). The interaction of HSF and TBP is explored in some detail here and is shown to be mediated by residues in both the amino- and carboxyl-terminal portions of HSF. This HSF/TBP interaction can be specifically disrupted by competition with the potent acidic transcriptional activator VP16. We further show that the acidic domain of the largest subunit of Drosophila RNA polymerase II (Pol II) associates with TBP in vitro and is specifically displaced from TBP upon addition of HSF. The region of TBP that mediates both HSF and Pol II acidic domain binding maps to the conserved carboxyl-terminal repeats and depends on at least one of the TBP residues known to be contacted by VP16 and to be critical for transcription activation. We discuss these findings in the context of a model in which HSF triggers hsp70 transcription by freeing the hsp70 promoter-paused Pol II from the constraints on elongation caused by the affinity of Pol II for general transcription factors.
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PMID:Cooperative and competitive protein interactions at the hsp70 promoter. 940 12

RNA polymerase II has been found to pause stably on several metazoan genes in a promoter-proximal region located 20-40 nt downstream from the start site of transcription. Escape of polymerase from this paused state has been proposed to be a rate limiting step in transcription of some genes. A study of the human hsp70 promoter showed that a nucleosome positioned downstream from the transcription start was a key component in establishing a stably paused polymerase in one cell-free system. We tested whether these results could be extended to the Drosophila hsp70 promoter in a Drosophila cell-free system and found that polymerase paused stably on the promoter even when the length of DNA downstream from the transcription start was not sufficient for assembly of a nucleosome. Our results indicate that a downstream nucleosome is not a universal requirement for stably pausing RNA polymerase in the promoter-proximal region.
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PMID:Nucleosomes are not necessary for promoter-proximal pausing in vitro on the Drosophila hsp70 promoter. 946 67

Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation.
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PMID:Transcriptional activation domains stimulate initiation and elongation at different times and via different residues. 960 96

The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.
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PMID:Human stress protein hsp70: overexpression in E coli, purification and characterization. 963 84

The effect of cell stresses upon the expression of the Bm1 short interspersed element (SINE) family in cultured silk worm cells is examined. Primer extension analysis shows that Bm1 repeats are transcribed by RNA polymerase III (Pol III) into cytoplasmic RNAs. Five consecutive T residues, which would normally terminate Pol III transcription, occur within the Bm1 consensus and are included within cDNA sequences representing these transcripts. In analogy to mammalian SINEs, the level of the Bm1 transcripts increases in response to either heat shock, inhibiting protein synthesis by cycloheximide or viral infection. The lifetime of Bm1 RNA increases following cell insults so that post-transcriptional events partially account for stress induced increases in its abundance. In the case of heat shock, the increase in Bm1 RNA follows the transient increase in hsp70 mRNA indicating that this response is temporally regulated to occur later in heat shock recovery. These results support the proposal that SINE RNAs serve a role in the cell stress response that predates the divergence of insects and mammals implying that SINEs are essentially a class of cell stress genes.
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PMID:Silk worm Bm1 SINE RNA increases following cellular insults. 1045 47


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