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)

A DNA-dependent RNA polymerase has been isolated from Caldariella acidophila, a thermophilic bacterium living in acidic hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme was purified 180-fold and is composed of five different subunits having the following molecular weights: a = 127000, b = 120000, c = 72000, d = 65000, and e = 38000. The enzyme is activated by Mn2+ and Mg2+ and exhibits optimal activity in the presence of 0.5 mM Mn2+. The activity depends on ionic strength, with a maximum at 0.25 M KCl, and exhibits a pH optimum at 7.8 in the presence of Tris-HCl buffer. The enzyme shows a high degree of thermophilicity, its temperature optimum being 80 degrees C in the in vitro assay. The thermophilicity of C. acidophila RNA polymerase allows studies on enzyme-template interactions to be performed in a temperature range where many templates are close to their Tm.
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PMID:DNA-dependent RNA polymerase from the thermophilic bacterium Caldariella acidophila. Purification and basic properties of the enzyme. 0 9

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

The putative viral transcriptase p90 in infectious bursal disease virus (IBDV) was shown to form an enzyme-guanylate intermediate which is indicative of guanylyl-transferase activity. The p90-nucleotide bond is most likely to be a phosphodiester linkage, as it resisted treatment with HCl and NH2OH but was sensitive to NaOH. This is in contrast to phosphoamide bonds formed by reovirus cores. Methyltransferase activity was also demonstrated in IBDV, and is closely associated with transcription, suggesting that p90 may be a multifunctional enzyme.
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PMID:Demonstration of enzyme activities required for cap structure formation in infectious bursal disease virus, a member of the birnavirus group. 215 6

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.
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PMID:Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica. 244 71

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. 354 18

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening. 390 Apr 14

When rat liver nuclear chromatin was sonicated in buffer containing 0.35 M (NH4)2SO4 to release the engaged RNA polymerases, a potent inhibitor was also released. This inhibitor elicited dramatic inhibition of RNA synthesis regardless of whether the free or engaged RNA polymerase was used. On further analysis, it became apparent that the site of inhibition was on the DNA template, not on the enzyme. This inhibitor could be extracted into 0.25 N HCl by the standard procedure for the isolation of histones. This acid-soluble inhibitor, showing typical histone band on gel, was RNase A and DNase I resistant, but was sensitive to both pronase and snake venom phosphodiesterase digestion, as well as to 0.1 N KOH hydrolysis. Furthermore, when [14C]adenine labeled poly-ADP-ribosylated histones were digested by snake venom phosphodiesterase, the release of radioactivity was in parallel to the loss of inhibitor activity. We conclude that the inhibitor substances are poly-ADP-ribosylated histones and propose that the poly-ADP-ribosylated histones rather than the histones are the natural suppressors of the gene.
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PMID:Poly-ADP-ribosylated histones: potent DNA suppressors. 404 88

Polyspermine-ribonuclease (Mr approximately 17 000) and the enzyme transcriptase from Rauscher-leukaemia virus (Mr approximately 70 000) form a complex Mr approx. 160 000) such that the molar ratio of polyspermine-ribonuclease to reverse transcriptase is 5:1. The most favourable condition for complex-formation is in a solution consisting of 0.01 M-Tris/HCl buffer, pH 7.5, 0.25 M-KCl and 1 mM-Mn2+ at 37 degrees C. The association of the two enzymes retains full RNAase activity, but reverse-transcriptase activity is completely inhibited when ribonuclease-sensitive polymers such as (dG)12 x (rC)n or viral 70S RNA are used as primer templates.
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PMID:Complexing reverse transcriptase with polyspermine-ribonuclease. 616 6

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) . (polycytidylic acid) even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.
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PMID:Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets. 628 Jun 8

Macronuclei of Paramecium aurelia, isolated in 4% gum arabic, contain nearly exclusively heterochromatin which is unaccessible to DNA-dependent, bacterial RNA polymerase. Heterochromatin decondensation under tris-HCl treatment did not change its template accessibility, whereas selective removal of the basic proteins induced profound changes in ultrastructure of heterochromatin and increased its template activity. More intensive incorporation of H3-UMP was found after arginine rich histones removal. The relations between functional and morphological changes after various heterochromatin modification in macronuclei of Paramecium aurelia, a representative of lower eukariotes, are discussed and compared with those in higher eukariotes.
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PMID:Ultrastructure and template accessibility of modified chromatin in isolated macronuclei of Paramecium aurelia. 699 78


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