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)

Nucleoli isolated from Novikoff hepatoma cells of the rat were previously shown to carry out synthesis of predominantly ribosomal precursor RNA and methylation of this RNA in vitro. In order to develop in vitro systems for further detailed study of these processes and their interrelationships, isolated nucleoli were incubated in a complete RNA-synthesizing medium using (5-3H)cytidine 5'-triphosphate or S-adenoxyl(methyl-3H)methionine to measure the activities of RNA synthesis and methylation, respectively, under the same reaction conditions. Methylation of the ribose of the nascent ribosomal precursor RNA predominated. It occurred in close coordination with the transcriptional step by RNA polymerase as shown by the kinetic data, the analysis of labeled RNA in sucrose gradients, the inhibition by increased ionic strength or actinomycin D, and the release of labeled nucleotides by a 3'-exonuclease, venom phosphodiesterase. Methylation of the RNA bases occurred more slowly, continued longer after transcription ceased, and appeared to follow later in the processing of the RNA. Certain divalent cations (Mg2+, Mn2+, and Ca2+ at higher concentrations, and Zn2+ and Cu2+) inhibited both RNA synthesis and methylation to similar extents. RNase inhibitors (bentonite and dextran sulfate) at low concentration inhibited methylation while stimulating RNA synthesis, and pyrophosphate greatly decreased RNA synthesis with relatively little effect on methylation. These results indicated that RNA polymerase and ribosomal RNA methylases can function independently despite their close relationship. An exogenous substrate for the nucleolar rRNA methylases was found: nuclear RNA prepared from Novikoff hepatoma cells, cultured in the absence of methionine, served as a good substrate for methylation of both ribose and bases. Other exogenous RNAs, including cytoplasmic ribosomal RNA from these methionine-starved cells, nucleolar RNA from normal cells, and wheat germ ribosomal RNA were almost devoid of methyl-acceptor activity. A description of these parameters helps establish isolated nucleoli as a suitable system for further study of interaction of RNA polymerase, methylases, and nucleases in control of synthesis of ribosomal RNA.
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PMID:Interrelationships between synthesis and methylation of ribosomal RNA in isolated Novikoff Tumor nucleoli. 16 25

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.
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PMID:RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures. 170 7

Proteins purified on the basis of their affinity for RNA polymerase II effectively substitute for previously defined transcription initiation factors. In two assays, formation of initiation complexes and transcription in vitro, the RNA polymerase II-associated proteins behaved identically to a fraction containing transcription factors IIE and IIF. Both fractions greatly stabilized the association of polymerase with the promoter and were required for the formation of complete initiation complexes. By using the DNA-cleaving reagent phenanthroline.copper in footprinting reactions, the RNA polymerase II-associated proteins were shown to be required for a DNA conformation change near the initiation site of the promoter. Based on similarity to the prokaryotic transcription complex, this conformation change is likely to represent a transition from a closed to an open complex.
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PMID:RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex. 188 89

The nuclease activity of the copper complex of 5-phenyl-1,10-phenanthroline (5-phi-OP-Cu) detects conformational changes in the lac UV-5 promoter caused by E. Coli RNA polymerase. The template strand in melted regions of initiation complexes upstream of the site of nucleotide triphosphate incorporation is very reactive. In open and elongation complexes, downstream scission sites (e.g. +4 and +5 for the open complex) on both strands are observed. The patterns of both downstream cutting sites suggest an atypical double helix at the leading edge of the transcription bubble.
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PMID:Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymerase. 218 56

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.
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PMID:Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. 284 43

Copper complexes of 1,10-phenanthroline (OP-Cu) hydrolyze DNA [D'Aurora, V., Stern, A. M., & Sigman, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 1025-1032; Marshall Pope, L., Reich, K. A., Graham, D. R., & Sigman, D. S. (1982) J. Biol. Chem. 257, 12121-12128]. This reaction has been studied to determine whether the 1,10-phenanthroline (OP) inhibition of the activity of RNA and DNA polymerases is the result of template hydrolysis or the chelation of a metal associated with and essential to the function of these enzymes. Addition of 4',6-diamino-2-phenylindole dihydrochloride (DAPI) to DNA generates a fluorescence signal with a linear increase of the intensity over a broad range of DNA concentrations from 0 to 100 micrograms/mL. The progress of hydrolysis of DNA by DNase I or OP (2 mM) is monitored by the time-dependent decrease in DAPI-induced fluorescence. In the presence of OP, the rate of hydrolysis increases as the Cu2+ concentration in the reaction mixture rises from 10(-8) to 10(-5) M. The rate differs for each nucleic acid template used; hydrolysis of poly(dA-dT) greater than denatured DNA greater than double-stranded DNA. However, millimolar amounts of OP do not hydrolyze the template even in the presence of Cu2+ (10(-6) M) when DNA is complexed with either Escherichia coli DNA polymerase I or Euglena gracilis or wheat germ RNA polymerase II. Under the same conditions, OP inhibits the activity of both varieties of RNA polymerase II with pKi's of 3.4 and 3.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of Euglena gracilis and wheat germ zinc RNA polymerases II by 1,10-phenanthroline acting as a chelating agent. 308 13

Metallothioneins that bind copper and zinc have an Mr of 6500 daltons, consist of a single polypeptide chain of 61 amino acids, 25-30 percent of whose residues are cysteine, have a metal-binding capacity of between 5 and 7 g atoms/mol, and contain no disulfide bonds or aromatic amino acids. Zincthionein has been postulated to participate in the transport and storage of zinc, which is involved in more than 235 metalloenzymes, including thymidine kinase, RNA polymerase, and ribonuclease, which in turn play crucial roles in the replication and transcription of DNA during cell division. In addition, trace elements including zinc modulate immune response and function. Conversely, zinc deficiency state causes, for example, thymic atrophy and lymphopenia and modifies antibody-mediated responses to both T-cell-dependent and T-cell-independent antigens. The concentrations of copper, zinc, and metallothionein and the copper/zinc ratio are modified in a number of malignancies. For example, the levels of metallothionein in normal and in malignant human livers are 471 and 75 micrograms/g, respectively. In addition, the copper/zinc ratio is significantly increased in human pancreatic cancer from 1.40 to 2.70. Furthermore, studies involving 64Cu in tumor-bearing mice showed that the distribution of 64Cu was altered and that all tumors contained a relatively high level of 64Cu. Moreover, the activity of superoxide dismutase to remove free oxygen radicals is lower in malignant tissues. Finally, the results of clinical studies suggest that the monitoring of the serum copper/zinc ratio may be a valuable tool, not only in determining the extent of malignancies, but also in predicting the efficacy of treatments.
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PMID:The status of zinc, copper, and metallothionein in cancer patients. 328 43

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.
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PMID:Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. 330 70

Protein-DNA complexes isolated in gel retardation assays can be digested within the acrylamide matrix by the nuclease activity of 1,10-phenanthroline-copper ion (OP-Cu). When the oligonucleotide products are eluted and analyzed on a sequencing gel, a footprint of the DNA-protein complex is obtained. Therefore, any protein-DNA complex isolated by the widely used gel retardation technique can be defined in terms of sequence-specific interactions by this simple methodology. The binding of the lac repressor and Escherichia coli RNA polymerase to an EcoRI fragment containing the lac control region has been studied by the combined gel retardation-1,10-phenanthroline-copper ion footprinting procedure. Footprints of lac repressor binding correspond to those obtained in solution with OP-Cu and DNase I and verify the experimental procedures. In studying E. coli RNA polymerase-promoter complexes, we have found that magnesium ion is required to form single-stranded DNA structures characteristic of kinetically competent open transcription complexes.
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PMID:Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. 332 97

Both the single DNA-dependent RNA polymerase found in zinc-deficient (-Zn) Euglena gracilis and the RNA polymerase III from zinc-sufficient (+Zn) cells have been isolated by methods previously used to purify polymerases I and II [Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1976) Biochemistry 15, 4468; Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1977) Biochem. Biophys. Res. Commun. 74, 1206]. Like class II polymerases, the enzyme from -Zn organisms elutes from DNA-cellulose and phosphocellulose with 0.6 M NaCl and 0.35 M NH4Cl, respectively. It is inhibited by 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, alpha,alpha'-bipyridyl, dipicolinic acid, and 1,10-phenanthroline (OP); 4,7-phenanthroline, the nonchelating analogue, does not inhibit. The pKI(OP) of this enzyme is identical with that of polymerase II but distinct from those of polymerases I and III. Elemental analysis confirms that zinc is the functional metal while copper, manganese, iron, and magnesium are absent. However, the -Zn enzyme is at least 4 orders of magnitude more resistant to alpha-amanitin (alpha-A) than the class II polymerase. Further, its response to alpha-A is unlike that of either polymerase I or polymerase III. Thus, -Zn cells contain a single, alpha-amanitin-resistant (alpha-Ar) RNA polymerase, whose behavior otherwise resembles that of the alpha-amanitin-sensitive polymerase II.
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PMID:Zinc deficiency and the Euglena gracilis chromatin: formation of an alpha-amanitin-resistant RNA polymerase II. 392 88


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