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)

Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n-d(A-T)n with adenosine 5'-[gamma-S]triphosphate and uridine 5'-[alpha-32P]triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogues did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel throught the entire transcript can be isolated.
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PMID:Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro. 33 43

We have employed mercury-substituted UTP to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. We find that the use of this method results in large overestimates of the amount of de novo synthesis of globin-specific RNA sequences. The artefact arises because endogenous globin RNA can serve as a template for the RNA polymerase, resulting in the formation of a duplex product in which one strand is the endogenous message, and the other is the mercury-labeled complementary strand. Subsequent purification of the mercury-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. We document the details of this mechanism and describe methods which will eliminate the artefact.
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PMID:Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates. 33 58

Mercurated uridine triphosphate has been used to label transcripts of chicken reticulocyte chromatin made with Escherichia coli RNA polymerase. The mercury-labeled RNA product can be completely separated from endogenous RNA sequences in the chromatin by passage through a sulfhydryl Sepharose column. Globin cDNA hybridization to the transcript shows that only 2.6 x 10-5 of the transcript is globin RNA. In contrast to this result, erythrocte chromatin transcript contains less than one tenth as many globin RNA sequences.
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PMID:In vitro transcription of chromatin in the presence of a mercurated nucleotide. 106 24

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).
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PMID:Bacterial resistances to inorganic mercury salts and organomercurials. 131 Nov 13

We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.
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PMID:A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. 133 70

Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes. Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.
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PMID:Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. 173 Dec 1

We have made site-specific mutations in the promoter-operator region of the mercury-resistance (mer) operon of transposon Tn501. Mutations were selected to alter the spacing between the -10 and -35 promoter elements without altering the sequence of a 7 bp dyad symmetrical sequence, which is the site of binding of the regulatory protein, MerR. (MerR acts as a repressor in the absence of mercuric salts and as an inducer in their presence, and binds to the same site in each case). Transcription from the mutant promoters was measured in vivo in the presence and absence of MerR and of mercuric salts; and the relative affinities of the mutant promoters for partially purified MerR were determined in vitro by gel-shift assay in the presence and absence of mercuric salts. The 19 bp spacer was found to be essential for correct induction and repression of the operon; a spacer size of 20 or 21 bp prevents induction, and a spacer size of 18 or 17 bp causes the promoter to be highly active under all conditions. Double mutations, which alter the position of the 7 bp dyad relative to the -10 and -35 sequences without altering their spacing prevent induction by the MerR-Hg(II) complex, demonstrating the tight constraints on the positions of MerR and RNA polymerase in the transcriptional complex. The data are compatible with a model for induction of the mer promoter involving a local conformational change in the DNA structure caused by the MerR-Hg(II) complex.
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PMID:Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. 216 6

Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion [Hg(II)] and activates it in the presence of Hg(II). In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD). Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR. Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry. Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex.
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PMID:Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter. 217 50

The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity. In addition, MerR exhibits a high degree of selectivity. Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required. The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively. This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism.
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PMID:Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. 218 94

A new affinity chromatographic procedure for the separation of transcriptionally active nucleosomes has been used to study the changes that take place in chromatin structure along the c-fos and c-myc genes when RNA synthesis is inhibited. Mercury-affinity chromatography separates the sulfhydryl-reactive nucleosomes of transcriptionally active genes from the compactly beaded, non-reactive nucleosomes of transcriptionally inert DNA sequences. The new procedure also discriminates between nucleosomes that have "unfolded" to reveal the previously shielded SH groups of histone H3 and nucleosomes that bind to the mercury column because of their association with thiol-containing non-histone proteins located in the transcription unit. Both classes of Hg-bound nucleosomes contain the c-fos and c-myc sequences, but only when they are being transcribed. We compared the effects of alpha-amanitin and actinomycin D on the transcription of c-fos and c-myc with the effects of each inhibitor on the distribution of the corresponding oncogenic DNA sequences in the chromatographically separated nucleosome fractions. It was found that the inhibition of RNA polymerase II by alpha-amanitin (added at the peaks of c-fos or c-myc expression in serum-stimulated BALB/c 3T3 cells) resulted in a rapid loss of affinity of the oncogene-containing nucleosomes for the mercury column. There was no corresponding effect on the mercury-binding properties of nucleosomes containing 28 S ribosomal gene sequences, which continue to be transcribed by amanitin-resistant RNA polymerase I. Therefore, the binding of the c-fos and c-myc nucleosomes to the mercury column seems to depend upon reversible structural changes associated with their transcription. Surprisingly, there was no corresponding loss of affinity of the c-fos and c-myc nucleosomes for the mercury column when actinomycin D was employed to inhibit RNA synthesis, despite the fact that transcription of both genes had been arrested abruptly. Measurements of [3H]actinomycin D binding show its preferential intercalation into the transcriptionally active nucleosomes. We suggest that the intercalation of actinomycin D into the DNA of active nucleosomes can lock the transcription complex into an "unfolded" but potentially active configuration. This was confirmed by run-off transcription assays showing a restoration of c-fos and c-myc RNA synthesis when actinomycin D was displaced by proflavine.
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PMID:Reversible and irreversible changes in nucleosome structure along the c-fos and c-myc oncogenes following inhibition of transcription. 232 30


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