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)

In Saccharomyces cerevisiae cells harboring a GAL1 promoter-linked beta-galactosidase gene, the simultaneous expression of Escherichia coli DNA topoisomerase I and inactivation of yeast DNA topoisomerases I and II reduces the cellular level of beta-galactosidase to an undetectable level. Analysis of intracellular mRNA level and the density of RNA polymerase along DNA indicates that this reduction is due to the suppression of transcription and that both plasmid-borne and chromosomally located genes are affected. These results are interpreted in terms of inhibition of transcription in vivo due to positive supercoiling of the DNA template: preferential removal of transcription-generated negative supercoils by E. coli DNA topoisomerase I in the absence of both yeast DNA topoisomerases I and II results in the accumulation of positive supercoils in intracellular DNA. In normal prokaryotic or eukaryotic cells, accumulation of positive supercoils is presumably avoided through the balanced actions of DNA topoisomerases.
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PMID:Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast. 133 10

In the yeast Saccharomyces cerevisiae, the ribosomal RNA genes are present in a single tandem array. A transcriptional enhancer element lies within the spacer region between each rRNA gene, 2.2 kilobases upstream from the transcription initiation site. We have identified previously two proteins, REB1 and REB2, that bind to specific sites within the enhancer (Morrow, B. E., Johnson, S. P., and Warner, J. R. (1989) J. Biol. Chem. 264, 9061-9068). REB1 binds also to a second, higher affinity site near the promoter, 210 base pairs upstream from the initiation site. This report describes the purification and further characterization of REB1. REB1 is a single polypeptide with an apparent molecular mass of 125,000 Da that binds to the sequence CCGGGTAA. It has been found to bind also within transcriptional control regions of several genes transcribed by RNA polymerase II, such as the UASG of the GAL1-GAL10 spacer. Immunoprecipitation analysis demonstrated that REB1 is phosphorylated.
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PMID:Purification and characterization of the yeast rDNA binding protein REB1. 224 86

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.
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PMID:Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. 240 41

A DNA fragment encompassing the Saccharomyces cerevisiae GAL1--GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase. We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA. Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand-specific endonuclease Pl. In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined. Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS). In vitro transcription with purified S. cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences.
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PMID:Structure of RNA polymerase II promoters. Conformational alterations and template properties of circularized Saccharomyces cerevisiae GAL1-GAL10 divergent promoters. 301 25

The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life approximately equal to 5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent.
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PMID:Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis. 302 Mar 64

We have constructed a yeast strain (UKY403) in which the sole histone H4 gene is under control of the GAL1 promoter. This allows the activation of H4 mRNA synthesis on galactose and its repression on glucose. UKY403 cells, pre-synchronized in G1 with alpha-mating factor, have been used to show that glucose treatment results in the loss of approximately half the chromosomal nucleosomes. This depletion is only partially reversible when the H4 gene is reactivated on galactose. It was found that the resultant lethality manifests itself first in S phase, the period of nucleosome assembly, but leads to highly synchronous arrest in G2 and a virtually complete block in chromosomal segregation. Histone H4-depleted chromatin was analyzed for its efficiency as a template for all three RNA polymerases. Using pulse-labeling, we find no evidence for altered transcription by RNA polymerase I (25S, 18S and 5.8S rRNAs) or RNA polymerase III (5S rRNA, tRNAs). Northern blot analysis was used to measure levels of RNA polymerase II transcripts. There was little effect on the activation or repression of the CUP1 chelatin gene. While there may be some decrease in the level of certain mRNAs (e.g. HIS4, ARG4) other message levels (HIS3, TRP1) show little change upon glucose repression. Therefore, nucleosome loss certainly does not have a general effect on transcription.
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PMID:Effects of histone H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae. 304 33

Gene activation by a DNA-binding regulatory protein in yeast requires the protein to have two components: one to recognize a specific DNA sequence and a second, the 'activating region', to interact with a general transcription factor or perhaps with RNA polymerase. The activating regions that have been characterized are acidic, and mutational analysis of one indicates that this acidity is important for activity. Here we report the design of an artificial protein bearing a novel 15-amino acid peptide linked to a DNA binding fragment of the yeast regulatory protein GAL4). The synthetic peptide is acidic and should it form an alpha-helix, that helix would be amphipathic, having one hydrophilic face bearing the acidic residues, and one hydrophobic face. When expressed in yeast, the artificial protein bearing this peptide efficiently activates the GAL1 gene which is ordinarily activated by GAL4. An otherwise identical protein with the novel 15 amino acids in a scrambled order, and which is thus unable to form an amphipathic structure, does not activate GAL1 transcription.
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PMID:Transcription in yeast activated by a putative amphipathic alpha helix linked to a DNA binding unit. 331 67

The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.
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PMID:Structure-function relationship of yeast S-II in terms of stimulation of RNA polymerase II, arrest relief, and suppression of 6-azauracil sensitivity. 772 9

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.
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PMID:Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle. 796 60

An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.
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PMID:T7 RNA polymerase-dependent expression of COXII in yeast mitochondria. 800 68


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