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)

Two types of particles were isolated during purification of rotavirus. Dense (D) particles have a density of 1.38 in CsCl and exhibit spontaneously a fully active endogenous transcriptase. Light (L) particles (density of 1.36 in CsCl) need to be treated with chelating agents to show a polymerase activity. The activation process of L particles was studied under strictly controlled monovalent, divalent, and hydrogen ion concentrations. These experiments demonstrate that i) activation is not affected by the ionic strength ii) activation occurs only at a pH higher than 7.1 iii) a low concentration of chelating agent (40 muM EDTA) is sufficient to activate the enzyme. Treatment of particles with EGTA, which chelates selectively Ca2+, leads to unmasking even in the presence of magnesium, indicating that the concentration of free calcium ions plays a major role in the activation process. Various glycosidases, detergents, and chelating agents were tested in respect to unmasking properties. Of these compound only chelating agents turned out to be efficient. Following activation, two glycopeptides were solubilized. These glycopeptides have an apparent molecular weight of 34,000 and 31,000 daltons and react with concanavalin A. The role of Ca2+ upon the stability of virus particles, and the activation of the endogenous transcriptase in vitro and in the infected cells is discussed.
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PMID:Activation of rotavirus RNA polymerase by calcium chelation. 4 4

The principle amatoxin, alpha-amanitin, is found to be extremely sensitive toward lactoperoxidase catalyzed degradation, rather than iodination, of the indole nucleus. Extensive attenuation of inhibitor potency against eukaryotic DNA-dependent RNA polymerase II accompanies the treatment of alpha-amanitin with lactoperoxidase, iodide and hydrogen peroxide.
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PMID:alpha-Amanitin: inactivation by bovine lactoperoxidase. 10 6

When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
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PMID:Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. 16 2

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.
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PMID:RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses. 18 17

A nucleoprotein complex that is an intermediate in viral transcription has been isolated from simian virus 40 (SV40)-infected BSC-1 cells after lysing infected nuclei with Sarkosyl. It contain DNA, DNA-dependent RNA polymerase II, and nascent RNA chains. RNA chain elongation continues for several hours in vitro and is dependent on exogenous ribonucleoside triphosphates. The complex sediments in neutral sucrose gradients with a main peak at about 24 to 26S. When the nascent RNA on the complex is treated with RNase A, a fraction of the RNA remains resistant to RNase and is hydrogen bonded to the DNA template. The pulse-labeled RNase-resistant RNA can be chased into RNase-sensitive RNA, indicating that it is located at the 3' terminus of the RNA chain. The rate of RNA displacement from the DNA template is consistent with an average rate of RNA chain elongation of 15 to 30 nucleotides per min. At least 70% of the RNA synthesized in this in vitro system is SV40 specific. Hybridization with the separated strands of SV40 DNA and with fragments of SV40 DNA generated with endonucleases HindII + III indicates that this RNA is complementary to all regions of the "late" SV40 DNA strand. Studies of SV40 RNA synthesis in this partially purified preparation at early and late times after infection should provide a way of locating promoter sites for transcription and identifying the form of SV40 DNA that serves as a template for late transcription.
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PMID:Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells. 19 3

Simian Virus (SV40) transcriptional intermediates (T.I.) were isolated from infected cell nuclei incubated in vitro in the presence of the four ribonucleoside triphosphates. The nascent mRNA strands in the viral DNA-RNA hybrid molecules were hydrogen bonded to their template by 200-250 nucleotides on the average, as judged from the extent of their RNase resistance and the aspect of T.I. under electron microscope after treatment with 50 per cent formamide. The RNA polymerase involved (RNA polymerase II) synthesized up to full length transcripts at a rate of approximately 150 nucleotides/min. at 25 degrees C. Each SV40 infected cell was found to contain about 200 active T.I. molecules at the peak of late transcription. The DNA in the T.I. molecules was exclusively form I DNA only in cell infected with the tsA30 mutant of SV40 that had been transferred to non-permissive temperature in order to arrest DNA replication, but both form I DNA and molecules behaving as replicative intermediates (R.I.) in wild type infected cells.
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PMID:Characterization of simian virus 40 transcriptional intermediates in infected CV1 cell nuclei. 23 Aug 57

The structure of premature core enzyme, an obligatory intermediate in both in vivo and in vitro assembly of Escherichia coli DNA-dependent RNA polymerase, was compared with that of native core enzyme. Though this assembled but inactive form of core enzyme harbors the gross conformation similar to that of native enzyme, minor and presumably local differences exist, which were identified by near-ultraviolet circular dichroism spectra, tritium-hydrogen exchange rate, protease sensitivity, intersubunit cross-linking rate by bifunctional reagents, sedimentation behavior, and elution profile from phosphocellulose. Taken together these results indicate that the core enzyme subunits are loosely associated in the premature core. The temperature-dependent maturation is required for the core subunits to be tightly associated, leading to the formation of structurally stable and functionally active RNA polymerase.
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PMID:Subunits of RNA polymerase in function and structure. 7. Structure of premature core enzyme. 37 70

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.
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PMID:Biochemistry of the amatoxins: preparation and characterization of a stably iodinated alpha-amanitin. 62 38

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

The inhibitory effect of the polypeptide antibiotics netropsin and distamycin A on DNA dependent nucleic acid synthesis has been shown to be related to the base composition of the template DNA. A number of natural DNA's of quite different dA-dT content as well as poly (dI-dC)-poly (dI-dC), poly (dA-dT)-poly (dA-dT), poly (dA) - poly (dT) and poly (dG) - poly (dC) has been studied as templates in DNA and in part in RNA polymerase reaction. The highest binding efficiency of netropsin existing for (dA-dT) - containing DNA polymers and the less pronounced interaction with the (dI-dC)-containing polymer shown by the melting and CD spectrral behaviour of the complexes are entirely reflected in the template inactivation. The same is evident for distamycin A. However, in contrast to netropsin the antibiotic distamycin A exhibits some binding tendency to poly (dG) - poly (dC). Binding effects of a netropsin derivative to DNA and (dA-dT) -containing polymers suggest the importance of hydrogen bonds of the peptide groups in the complex formation.
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PMID:(dA-dT) dependent inactivation of the DNA template properties by interaction with netropsin and distamycin A. 109 41


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