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)

We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [3H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [3H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [3H]uridine by greater than 75%. Cellular uptake of [3H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [3H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.
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PMID:Mechanism of apparent transcription inhibition by methyl mercury in cerebellar neurons. 242 3

Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.
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PMID:The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercury on RNA synthesis in isolated nuclei. 244 20

An assay that employs guanosine 5'-O-(2-thiotriphosphate) was used to measure correct initiation of RNA chains in isolated cell nuclei, where chromatin structure is relatively undisturbed. RNA chains initiated with guanosine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated mouse mammary tumor virus RNA with a T1 nuclease protection assay. The monovalent cation concentration dependence for initiation in isolated nuclei was similar to that previously observed for initiation from naked DNA templates (optimum near 90 mM) but different from that for elongation of nascent RNA chains. However, in contrast to the systems that employ naked DNA templates, initiation efficiency in the nuclear system was relatively unaffected by moderate changes in pH (6.7-8.3), temperature (25-37 degrees C), and magnesium ion concentration (1-9 mM). The optimized assay was used to assess the inhibitory activity of several compounds that have been reported to be specific inhibitors of transcription initiation on naked DNA templates. Both Sarkosyl and heparin were effective inhibitors of specific initiation by RNA polymerases I and II in isolated nuclei without inhibiting elongation of nascent chains, but 5,6-dichlorobenzimidazole riboside was relatively ineffective as a specific inhibitor of initiation by RNA polymerase II.
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PMID:Optimized reaction conditions and specific inhibitors for initiation of transcription by RNA polymerase II in nuclei from cultured mammalian cells. 244 43

Methyl mercury (MeHg) inhibited the overall RNA synthetic reaction of HeLa RNA polymerase II. However, when RNA chain initiation was allowed to occur in its absence, MeHg stimulated the rate of the subsequent elongation stage of the reaction. Chain elongation with both double-stranded and single-stranded DNA templates was stimulated. This stimulatory effect was specific for MeHg; both p-hydroxymercuribenzoate and HgCl2 inhibited chain elongation (to about the same degree as they inhibited the overall reaction). The stimulatory effect was also specific for the HeLa polymerase; with Escherichia coli RNA polymerase, MeHg inhibited elongation (to the same degree as it inhibited the overall reaction).
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PMID:Methyl mercury stimulates chain elongation by purified HeLa RNA polymerase II. 246 8

Double-stranded DNA which was exposed to methyl mercury at concentrations of 1 mM and above, and purified by ethanol precipitation and dialysis, was transcribed at a higher rate by RNA polymerase II than was control DNA. The rate of transcription of single-stranded DNA was not affected by similar exposure to methyl mercury. The higher rate of transcription of methyl mercury-treated double-stranded DNA appears to result from a decreased Km of the enzyme for this DNA. This does not appear to result from extensive denaturation, nor from formation of a large number of single-stranded breaks in the DNA.
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PMID:Exposure of DNA to methyl mercury results in an increase in the rate of its transcription by RNA polymerase II. 258 May 21

Expression of the mercury-resistance (mer) genes of the transposon Tn501 is positively and negatively controlled by the product of the merR gene. DNA sequence analysis has identified three open reading frames as potential candidates for this gene, one of which is oriented divergently with respect to the mercury-resistance genes. We have demonstrated that although RNA polymerase will bind to fragments containing the potential control regions for all three reading frames, only the control region for this divergent reading frame shows detectable promoter activity in vivo. Transcription of this reading frame is required for repression and induction of mer transcription. We have also shown that the Tn501 merR gene product negatively regulates its own synthesis, and have identified the start point of the transcript for this reading frame and for the mercury-inducible transcript of the mercury-resistance genes.
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PMID:Transcriptional regulation of the mercury-resistance genes of transposon Tn501. 301 64

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.
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PMID:Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21). 303 86

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.
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PMID:RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. 309 67

A previous publication [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)] described the observation that double-stranded DNA which was briefly exposed to methyl mercury (MeHg) and purified to remove free methyl mercury was transcribed at a higher rate by RNA polymerase II from wheat germ. The specificity of this phenomenon has now been investigated by examining the activity of this MeHg-exposed DNA as a template-primer for DNA polymerases. DNA synthesis by the bacteriophage T4-induced DNA polymerase was higher with the MeHg-exposed DNA as a template-primer than with control DNA. In contrast, the rate of DNA synthesis by E. coli DNA polymerase I was lower with the MeHg-exposed DNA as template-primer. With both enzymes (as well as with RNA polymerase II), after denaturation of the MeHg-exposed and control DNAs the differences in template activity were either eliminated or markedly reduced. The enzymes are thus able to detect a MeHg-induced alteration in DNA. In contrast, circular dichroism, a physical method that is sensitive to conformational changes in DNA, did not detect any difference between the MeHg-exposed and control DNAs.
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PMID:Effects of exposure of DNA to methyl mercury on its activity as a template-primer for DNA polymerases. 352 50

As part of a long-term cytogenetic research project on mercury, we studied the in vitro clastogenic capacity of HgCl2 and CH3HgCl as well as their influence on chromosome segregation by means of a computer-aided chromosome distribution study in metaphase plates. As in other in vitro studies published elsewhere, we exposed human peripheral blood lymphocytes to different concentrations of the mercury compound during a limited period of the pre-DNA synthetic stage (G1-S) or from that stage up to mitosis (G1-M). For both exposure periods and both mercury compounds we observed a rather important clastogenic effect as well as a dissociation of the (normally highly associated) acrocentrics. The results do indicate, in conjunction with previously published data, that mercury compounds alter the chromosome segregation at lower concentrations than those observed for clastogenicity. Moreover, the effects on chromosome segregation are not necessarily due to binding to spindle proteins. Binding to--and inactivation of RNA polymerase I may for example be another mechanism of action which is more important for the inorganic form of mercury than for the organic form.
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PMID:Comparative in vitro cytogenetic studies in mercury-exposed human lymphocytes. 402 43


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