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 activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.
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PMID:Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo. 191 50

A 66 base-pair (bp) DNA template carrying a site-specifically placed psoralen cross-link downstream from a phage T7 promoter was constructed. This template can support transcription by T7 RNA polymerase. Transcription was blocked specifically at the psoralen cross-link. We studied the characteristics of elongation complexes, formed in this manner, by enzymatic and chemical footprinting and by a nitrocellulose filter-binding assay. The DNase I footprint of the elongation complex was quantified on a residue by residue basis. It was found that T7 RNA polymerase made the strongest contacts in the central region of the footprint whereas the leading and lagging edges of the polymerase were weakly bound to the DNA. Reducing the NaCl concentration in the transcription reaction resulted in the visualization of two T7 RNA polymerase molecules bound to the same template. A leading polymerase molecule, arrested at the psoralen cross-link, showed a much smaller DNase I footprint than a lagging polymerase molecule that was bound upstream. This upstream polymerase molecule occupied approximately one-half of the promoter region and therefore did not achieve complete promoter clearance. These experiments suggest that complete promoter clearance is required for a gross conformational change in the polymerase, consisting of a contraction in the size of its footprint to occur. DNase I footprinting also revealed that an elongation complex arrested at a psoralen cross-link undergoes several subtle changes in structure in a time-dependent manner and therefore can be considered to be in a state of dynamic flux. Methylation protection showed that some G residues in the top (non-coding) strand are protected against attack by dimethylsulfate, whereas the G residues on the bottom (coding) strand appear not to be protected from reaction with dimethylsulfate. We probed the transcribing complexes for single-stranded regions with T7 gene 3 endonuclease. From the pattern of sensitivity to T7 gene 3 endonuclease on the template strand, we conclude that the RNA-DNA hybrid in the elongation complex is about 7 bp. A nitrocellulose filter-binding assay showed that the elongation complex, consisting of a 36 (+1) nucleotide RNA, the 66 bp DNA template and the T7 RNA polymerase was stable for at least 30 minutes at high salt concentrations. Heparin caused the quantitative release of 36 (+1) RNA nucleotides within 30 seconds, but the DNA was not simultaneously released from the elongation complex under these conditions.
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PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes. 194 44

We constructed a 66 base-pair DNA template capable of supporting transcription by T7 RNA polymerase. This template had a psoralen cross-link downstream from a T7 promoter such that a 36 (+1) nucleotide transcript was synthesized at the time the T7 polymerase came to a stop at the cross-link. The stability of elongation complexes formed on this template, and the effect of different factors that are known to affect polymerase-DNA interactions was investigated by non-denaturing gel electrophoresis and gel filtration chromatography. We found that an elongation complex could lose its RNA component but the T7 polymerase still remained attached to the DNA template for extended periods of time (at least up to 18 h). This type of an elongation complex, bereft of its nascent RNA transcript, is called a quasi-elongation complex. DNase I footprinting within gel slices indicated that the polymerase molecules were arrested at the psoralen cross-link on the DNA template in the quasi-elongation complexes. The quasi-elongation complexes were found to be extremely stable in 0.5 M-NaCl and in 0.2 M-NaCl plus 60 mM-MgCl2, and could withstand temperatures up to 42 degrees C. The quasi-elongation complexes were destabilized by heparin and excess calf thymus DNA. Excess tRNA caused only a minimal degree of disruption. Non-promoter-containing plasmid DNAs did not have a destabilizing effect on the quasi-elongation complexes. Interestingly, it was observed that in a T7 ternary transcriptional complex arrested by a psoralen cross-link, the nascent RNA transcript could be stabilized from release by the presence in trans of a plasmid DNA bearing a T7 promoter and a T7 terminator. Such a stabilization against RNA release was not observed with plasmid DNAs containing either only a promoter or a terminator. The elongation complexes were stable during gel filtration through Sephacryl S-300 HR. However, it was found that 30% to 45% of the labeled RNA was retained during gel filtration as RNA that was apparently free from ternary complexes.
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PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. II. Stability and some properties of elongation complexes. 194 45

During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.
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PMID:Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes. 194 86

Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture. This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA). Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source. Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes. We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.
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PMID:High yield purification of active transcription factor IIIA expressed in E. coli. 195 78

The promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli gene aroG (encoding the phenylalanine-sensitive 3-deoxy-arabinoheptulosonate-7-phosphate synthase) were located. Primer extension analysis and nuclease S1 mapping of in vivo transcripts were used to determine the 5' and 3' ends, respectively, of the mRNA. Both ends exhibited some heterogeneity with respect to length. The 3' end of the major molecular species was located within a region that has structural homology with known rho-independent terminators. The location of the aroG promoter was identified in both strands of the DNA by in vitro DNase I footprinting and methylation protection experiments with RNA polymerase. In these experiments, a region of up to 80 base pairs (bp) was protected by the binding of RNA polymerase. The location of the aroG operator was also identified in both strands of the DNA by in vitro DNase I footprinting with pure TyrR. TyrR protected 26 to 28 bp of DNA containing a 22-bp palindrome (TYR R box) and overlapping the -35 region of the promoter. Mutations in the aroG regulatory DNA were isolated by site-directed mutagenesis and cloned in a low-copy-number plasmid to generate aroG-lac fusions. The effects of the mutations on the regulation of aroG expression were determined by measuring the beta-galactosidase activities of the fusions in strains with tyrR, tyrR+, and multicopy tyrR+ genotypes. The results of this mutant analysis confirmed that the aroG operator contains a single TYR R box.
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PMID:Identification of the promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG. 197 May 63

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
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PMID:Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change. 198 53

Utilizing yeast strains containing promoter mutations, we demonstrate that transcription of the HSP82 gene causes nucleosomes toward the 3'-end to become DNase I sensitive and 'split' into structures that exhibit a 'half-nucleosomal' cleavage periodicity. Splitting occurs even when only a few RNA polymerase II molecules are engaged in basal level transcription or during the first round of induced transcription. The split nucleosomal structure survives nuclear isolation suggesting that it may be stabilized by post-translational modifications or non-histone proteins, and may require DNA replication for reversal to a whole nucleosomal structure. Split nucleosomes represent a structure for DNase I sensitive chromatin and are probably of common occurrence but difficult to detect experimentally. We suggest that transient positive supercoils downstream of traversing RNA polymerase lead to nucleosome splitting.
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PMID:Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin. 200 76

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233-2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one-step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the -35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl-CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.
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PMID:Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor. 200 80

Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL-encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)-containing form of RNA polymerase. This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons. In vivo monitoring of the activity of a Pu-lacZ fusion in E. coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA-binding domain of the XylR does not impair its inhibitory effect when hyperproduced. One discrete IHF binding site and two potential XylR sites (consensus sequence 5'-TTGANCAAATC-3'), bracketted by short stretches of DNase I-hypersensitive bonds, were detected upstream of the transcription initiation site. A model accounting for the features found is proposed which includes the IHF-promoted looping of upstream XylR-DNA complexes so that they contact the sigma 54(NtrA)-RNA polymerase bound at -12/-24 positions.
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PMID:An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. 202 86


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