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)

Highly purified preparations of the DNA-dependent RNA polymerase obtained from Escherichia coli contain about 2 g-atoms of tightly bound zinc per mol (molecular weight 370,000) of enzyme. When the purified enzyme is fractionated on Sephadex G-150 or G-200, correlation is observed between the zinc and enzymic activity. Although some of the preparations examined also contain iron, copper, and magnesium, the content of these metal ions show no consistent correlation with RNA polymerase activity. Initiation of RNA synthesis is specifically inhibited by 1,10-phenanthroline. Less-effective inhibition is observed for other chelating agents or for a nonchelating phenanthroline analog. The analog also exhibits a pattern of inhibition differing from that characteristic of 1,10-phenanthroline. Binding of purine nucleoside triphosphates at the lower-affinity (K(d) = 0.15 mM) site may also be prevented by the addition of 1,10-phenanthroline. One or both of the bound zinc atoms may, therefore, participate in the initiation of RNA synthesis.
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PMID:The presence and possible role of zinc in RNA polymerase obtained from Escherichia coli. 494 29

1. The effects of various ions on the Mg(2+)- and Mn(2+)/ammonium sulphate-activated RNA polymerase activities of isolated liver nuclei were studied. 2. The Mg(2+)-activated RNA polymerase reaction was inhibited by more than 60% by Cd(2+), SeO(3) (2-), Be(2+), Cu(2+), Co(2+), Ca(2+) and La(3+), all at 1mm concentrations. 3. The Mn(2+)/ammonium sulphate-activated RNA polymerase reaction was strongly inhibited by Hg(2+), Cd(2+), Cu(2+) and Ag(+). The effect of Hg(2+), Cd(2+) and Ag(+) was relieved by cysteine or mercaptoethanol. 4. Inhibition by Cu(2+) was not affected by addition of DNA, and was relieved only partially by EDTA or histidine. 5. No changes of RNA polymerase activities were observed in nuclei isolated from the liver of rats treated with copper albuminate.
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PMID:The effects of copper and other ions on the ribonucleic acid polymerase activity of isolated rat liver nuclei. 497 30

The effects of 14 metal ions (chlorides) on the transcription of calf thymus DNA and phage T4 DNA with Escherichia coli RNA polymerase were tested. These assays were conducted under improved conditions of lower pH and in the absence of 2-mercaptoethanol to permit greater stability of the metal ions in solution. Among the divalent metal ions tested, the concentration-dependent order of inhibition of overall transcription is Pb2+ greater than Zn2+ greater than Cu2+ greater than Be2+ greater than Cd2+ greater than Ni2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Sr2+ and is the same with either template. At pH 7.4 and in the absence of 2-mercaptoethanol, considerably lower concentrations of several of the divalent metal ions are needed for inhibition of overall transcription than at pH 8.1 and in the presence of 2-mercaptoethanol. Ca2+, Mg2+, Sr2+, Zn2+, Li+, Na+, and K+--considered to be non-mutagenic and non-carcinogenic--decrease chain initiation (measured with T4 DNA) at concentrations that inhibit overall transcription. Pb2+, Cd2+, Co2+, Be2+, and Mn2+--all mutagenic or carcinogenic--stimulate chain initiation (although at widely different rates) at concentrations that inhibit overall transcription. Cu2+ and Ni2+--both carcinogenic--stimulate initiation only at very low concentrations, followed by a progressive decrease in initiation at concentrations that inhibit overall transcription.
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PMID:Selective effects of metal ions on RNA synthesis rates. 617 51

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.
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PMID:Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide. 627 85

By use of poly(dA-dT) as template and Escherichia coli RNA polymerase, several metal ions were tested for their effect on the efficiency of transcription and on the misincorporation of CMP into the poly(rA-rU) product. In the presence of 10 mM MgCl2, Mn2+ has a stimulatory effect on the transcription, Co2+ has very little effect on the reaction, Cu2+ and Zn2+ are strongly inhibitory, and Cd2+ and Ni2+ are less inhibitory. The background misincorporation of CMP in the presence of MgCl2 is about 1 nucleotide per 2000 correct nucleotides incorporated and is independent of Mg2+ concentration. Zn2+, Ca2+, Sr2+, Li+, Na+, and K+--all nonmutagenic and noncarcinogenic--do not increase misincorporation. Mn2+ causes a concentration-dependent threefold increase in the misincorporation that can be slightly reversed at higher MgCl2 concentrations. Cd2+ causes a dramatic increase in the misincorporation with increasing CdCl2 concentration that can be substantially overcome by higher concentrations of Mg2+. Cu2+ also increases the misincorporation, Ni2+ slightly increases it, and Co2+ does not increase it at all. Several control experiments indicate that the misincorporation of CMP is dependent on the template-directed synthesis of poly(rA-rU). Nearest-neighbor analysis indicates that CMP is incorporated in place of UMP into the poly(rA-rU) product. The increase in misincorporation appears to be related both to the "hard-soft" character of the metal ions and to their carcinogenic potential.
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PMID:Effect of several metal ions on misincorporation during transcription. 702 4

tRNAPhe transcribed in a T7 RNA polymerase system has been modified in such a way that 4-thiouridines have randomly replaced unmodified uridines. These 4-thiouridines serve as sites for conjugation of the cleavage reagent 5-iodoacetamido-1,10-phenanthroline (IOP). 1,10-Phenantholine, when complexed with Cu2+ in a reducing environment, causes hydrolysis of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-OP) conjugates, when bound in situ to the P- and E-sites of 70 S ribosomes, cause cleavage, mainly in domains I, III and V of 23 S ribosomal RNA (rRNA). The cleavage sites in domain V predominantly occur very close to or in the peptidyl-transferase region. The regions of domain I and III that are cleaved are apparently folded in the 50 S ribosomal subunit so as to be proximal to the peptidyl-transferase center. Most of the cleavage events occur whether the tRNA-OP conjugate is bound to ribosomes alone, or yeast tRNA is also present in the P/P hybrid state. Cleavages that occur only in the absence of yeast tRNA are limited to the 1100 region of domain II, and the 2800 region of domain VI. Cleavages that occur only in the presence of yeast occur in the 2170 region of domain V. The regions of 23 S rRNA in which tRNA-OP induced cleavage occur complement those sites shown by chemical protection and cross-liking to be in a close proximity to the tRNA. However, the cleavage approach allows a more versatile and expanded view of the near neighborhood of rRNA surrounding the tRNA. These results provide considerable information which will allow a more detailed modeling of the tertiary structure of the 50 S ribosomal subunit.
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PMID:Regions of 23 S ribosomal RNA proximal to transfer RNA bound at the P and E sites. 756 75

In the Escherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E. coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.
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PMID:Improvement of Escherichia coli cell-free system by utilization of cell extract having additional property. Problems and countermeasures. 762 23

An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase. Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide. When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed. The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies.
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PMID:Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex. Potential for gene-specific inactivation reagents. 814 87

The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein. We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate. Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA. Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex. We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex. Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.
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PMID:DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes. 815 88

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.
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PMID:A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes. 836 75


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