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 MeR regulatory protein of transposon Tn501 controls the expression of the mercury resistance (mer) genes in response to the concentration of mercuric ions. MerR is unique among prokaryotic regulatory proteins so far described in that it acts as a repressor [-Hg(II)] and an activator [+Hg(II)] of transcription of the mer genes, but binds to a single site on the DNA in both cases. This transcriptional activation process has been postulated to involve a protein-induced conformational change in the DNA that allows RNA polymerase more readily to form an open complex at the promoter. It has been shown [Frantz and O'Halloran (1990) Biochemistry, 29, 4747-4751] that activation of transcription by MerR in the presence of mercury is accompanied by hypersensitivity of the operator to chemical nucleases that are sensitive to local distortion in DNA structure. Here we describe specific mutations in MerR that allow the protein to stimulate transcription in the absence of the allosteric activator Hg(II). We demonstrate that the degree of activation caused by these mutants directly correlates with the degree of DNA distortion as measured by the hypersensitivity of MerR-DNA complexes to the nuclease Cu-5-phenyl-o-phenanthroline. These results support the model described above.
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PMID:Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg(II) is accompanied by DNA distortion. 844 Feb 34

The mercury resistance (mer) operon is transcribed from overlapping, divergent promoters: PR for the regulatory gene merR and P(TPCAD) for the structural genes merTPCAD. The dyadic binding site for MerR lies within the 19-bp spacer of the sigma70-dependent P(TPCAD). Unlike typical repressors, MerR does not exclude RNA polymerase from P(TPCAD) but rather forms an inactive complex with RNA polymerase at P(TPCAD) prior to addition of the inducer, the mercuric ion Hg(II). In this "active repression" complex, MerR prevents transcriptional initiation at merTPCAD until Hg(II) is added. When Hg(II) is added, MerR remains bound to the same position and activates transcription of merTPCAD by distorting the DNA of the spacer region. MerR also represses its own transcription from PR regardless of the presence or absence of Hg(II). To explore the role of MerR-RNA polymerase in these processes, we examined mutations in the sigma70 and alpha subunits of RNA polymerase, mutations known to influence other activators but not to impair transcription generally. We assessed the effects of these sigma70 and alpha mutants on unregulated P(TPCAD) and PR transcription (i.e., MerR-independent transcription) and on the two MerR-dependent processes: repression of P(TPCAD) and of PR and Hg(ll)-induced activation of P(TPCAD). Among the MerR-independent effects, we found that mutations in regions 2.1 and 4.2 of rpoD suppress the deleterious effects of nonoptimal promoter spacing. Some C-terminal rpoA mutants also have this property to a considerably lesser degree. Certain "spacer suppressor" variants of rpoA and of rpoD also interfere with the MerR-dependent repression of P(TPCAD) and PR. MerR-Hg(II)-mediated transcriptional activation of P(TPCAD) was also affected in an allele-specific manner by substitutions at position 596 of sigma70 and at positions 311 and 323 of alpha. Thus, certain changes in sigma70 or alpha render them either more or less effective in participating in the topologically novel transcriptional control effected by MerR at the divergent mer operons.
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PMID:Mutations in the alpha and sigma-70 subunits of RNA polymerase affect expression of the mer operon. 904 42

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.
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PMID:Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep. 921 Dec 28

MerR, the metalloregulator of the mercury resistance (mer) operon, binds the operator (merO)between -10 and -35 of the merTPCAD promoter (PT) and sequesters RNA polymerase (RNAP) in a closed complex. MerR represses PT until Hg(II) induces it to underwind merO DNA and thus facilitate open complex formation. We used cross-linking to determine if direct contacts between MerR and RNAP also occur during this process. MerR cross-linked to the alpha, beta, and sigma70 subunits of RNAP alone, indicating stable contacts which were further stabilized upon forming the preinitiation complex at PT. Hg(II) did not eliminate any of the MerR-RNAP cross-links but did increase the relative abundance of a MerR dimer conformer. Interference by MerR with self-cross-links among RNAP subunits and the formation of an electrophoretically stable association between MerR and RNAP also indicated MerR-RNAP interactions. This is the first evidence for stable physical contacts between MerR and RNAP and for a Hg(II)-induced allosteric change in MerR in the transcription-competent complex.
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PMID:MerR cross-links to the alpha, beta, and sigma 70 subunits of RNA polymerase in the preinitiation complex at the merTPCAD promoter. 1007 80

Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by sigma70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1, 000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR's metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.
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PMID:Cd(II)-responsive and constitutive mutants implicate a novel domain in MerR. 1034 59

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K(D)) for binding of MerR were: binding site I, 8.5 x 10(-9) M; binding site II, 1.2 x 10(-8) M; and for the complete promoter/operator region 1 x 10(-8) M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K(D) value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 x 10(-7) M.
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PMID:Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326. 1050 47

We report the discovery and characterization of the mercury resistance transposon, Tn5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn5044 is expressed at low (30 degrees C) but not at elevated temperatures (37 degrees C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn5044 is closely related to those of Tn1412 isolated from medical sources and to Tn5563 and ISXc5 from environmental sources. However, Tn5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase (tnpA) genes places Tn5044 and its close relatives into the Tn3 subgroup of the Tn3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn5044 resolvase can resolve cointegrates formed during Tn5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.
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PMID:Tn5044, a novel Tn3 family transposon coding for temperature-sensitive mercury resistance. 1087 86

Current evidence suggests that the nucleolus is composed of different substructures that are dynamic and form in response to the requirement for new ribosome synthesis. Thus, agents that disrupt nucleolar organization may deregulate basic cellular events and eventually contribute to human disease. Here we report that environmentally relevant concentrations (5 microM) of inorganic mercury induce a redistribution of nucleolar protein fibrillarin from the nucleolus to the nucleoplasm in epithelial cell lines. Since treatment with transcription inhibitors led to a similar relocation of fibrillarin, the effects of mercury on transcription were studied by run-on transcription assays: mercuric ions specifically blocked synthesis of ribosomal RNA, whereas activity of RNA polymerase II remained unchanged and occurred throughout the nucleoplasm. Moreover, we show by double-labeling that inhibition of nucleolar transcription and redistribution of fibrillarin occur simultaneously, underlining that fibrillarin relocation is a consequence of the blockade of ribosomal RNA synthesis by mercury. We also detected redistribution of fibrillarin in vivo, e.g., in splenic cells of mice chronically exposed to HgCl(2). Thus, implications of this alteration of nuclear structure and function for mercury-induced autoimmunity are discussed.
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PMID:Specific inhibition of rRNA transcription and dynamic relocation of fibrillarin induced by mercury. 1094 94

Research on autoimmune diseases has revealed that autoimmunity can be induced by heavy metals such as mercury and gold. Following the introduction of platinum-containing catalytic converters in automobiles, the emission of platinum compounds constitutes an abundant environmental pollutant, however, potential immunological hazards resulting from platinum-containing emissions were not yet examined. In our previous studies on molecular mechanisms of heavy metal-induced autoimmunity, we showed a platinum-dependent subcellular redistribution of the autoantigen fibrillarin from the nucleolus to the nucleoplasm. Since H-2s mice constitute a valuable model to study the role of heavy metals in the development of systemic autoimmunity, we treated susceptible B10.S mice with hexachloroplatinate (Na2PtCl6, Pt4+) to examine whether platinum induces the production of autoantibodies. The present study shows for the first time that chronic administration of Pt4+ generated an autoimmune response in mice which targets distinct nucleoplasmic antigens. Dual-labeling revealed substantial colocalization of these nucleoplasmic autoantigens with (i) nascent RNA, (ii) the active, phosphorylated form of RNA polymerase II, and partial overlap with (iii) acetylated histone 4 protein, and (iv) 20S proteasomes in dendritic cells isolated from platinum-treated mice. The results suggest that platinum elicits antibodies against antigens associated with active sites of transcription which may be subject to proteasomal processing during heavy metal-induced autoimmunity.
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PMID:Platinum-induced autoantibodies target nucleoplasmic antigens related to active transcription. 1260 22

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.
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PMID:[Distribution of transposons Tn5044 and Tn5070 with unusual mer operons in environmental bacterial populations]. 1564 57


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