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)

Labeling of RNA in isolated HeLa cell nuclei in vitro reveals an abundance of short RNA chains made by RNA polymerase II. These short chains were initiated prior to isolation of the nuclei. The short abundant chains are increased in amount in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Kinetic evidence indicates that the bulk of the putative heterogeneous nuclear RNA (hnRNA) precursor molecules that are terminated early in vivo are terminated approximately 100-300 nucleotides from sites of initiation. DRB increases the frequency of early termination, but there is a fraction of hnRNA precursor molecules whose elongation is not affected by DRB. Heparin is useful in studies of hnRNA transcription in isolated nuclei because it enhances chain elongation.
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PMID:Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole. 29 79

The kinetics of dissociation of the fd DNA - RNA-polymerase complex has been analyzed. Heparin was added to a solution of the enzyme - DNA complex in order to trap free polymerases. At different times after, samples were taken and analyzed by electron microscopy to determine the mean number of enzymes bound per DNA molecule. Unexpectedly, the measured dissociation is not a first-order reaction. The apparent rate constant increases with heparin concentration in the range between 0.001 and 2 mg/ml. These results strongly suggest the existence of a direct transfer process of RNA polymerase to heparin, bypassing the rate-limiting step of dissociation of the enzyme - DNA complex to free enzyme. Theoretical analysis of the direct-transfer model shows that the rate constant of dissociation should level off at high heparin concentrations: measurements of the residual transcription activity show that this is the case. From these experiments, the equilibrium constant of the DNA - RNA-polymerase complex can be determined. The value K = 10(12) M-1 which is obtained solves a striking paradox which existed because measurements performed in other laboratories indicated K = 10(14) M-1, which is greater than the equilibrium constant of the lac-repressor - lac-operator complex (=10(13) M-1).
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PMID:Electron microscopy analysis of the interaction between Escherichia coli DNA-dependent RNA polymerase and the replicative form of phage fd DNA. 2. Analysis of the dissociation kinetics. 33 32

Optimal conditions for isolation of rooster liver nuclei were studied for the purification of RNA polymerases I and II by varying the concentrations of sucrose, magnesium chloride and spermine in the isolation medium. Addition of spermine and/or magnesium chloride to the hypertonic sucrose medium was found to be advantageous for the purification. Heparin-Sepharose chromatography can be recommended for purification of RNA polymerases when used in a stepwise manner. Furthermore, gradient elution in the heparin-Sepharose chromatography was found to separate RNA polymerases I and II. RNA polymerase III was eluted with RNA polymerase I. A few minor peaks of RNA polymerase II activity were detected with gradient elution. Factors influencing the affinity of RNA polymerases towards heparin-Sepharose are discussed.
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PMID:RNA polymerases from avian liver. Isolation and fractionation by heparin-sepharose chromatography. 51 13

A transcriptionally active chromosome has been isolated in highly purified form from choroplasts of Euglena gracilis, It contains chloroplast DNA, DNA-dependent RNA polymerase, and other proteins. Transcription occurs at low levels of endogenous DNA, and is indifferent to high levels of exogenous DNA. RNA chain elongation continues for several hours in vitro, and RNA chain initiation, determined by [gamma-32P]ATP incorporation, is continuous for at least 1 h in vitro. Maximal rates for RNA synthesis require only a divalent cation and the four ribonucleoside triphosphates. Apparent Km values for adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate are 4.0, 0.6, 2.5, and 2.3 muM, respectively. As would be expected for a DNA-dependent RNA polymerase, RNA synthesis is inhibited by actinomycin D. However, rifampicin and streptolydigin, inhibitors of procaryotic RNA synthesis, and alpha-amanitin, an inhibitor of eucaryotic nuclear RNA polymerases II and III, do not inhibt the RNA synthesis reaction. Heparin, which is a potent inhibitor of the initiation of RNA synthesis by a nontemplate bound RNA polymerase, also does not inhibit RNA synthesis. Isolation of transcriptionally active chromosomes should prove to be a useful method to study the mechanism of selective RNA transcription of eucaryotic chromosomes.
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PMID:Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis. 82 16

Total RNA polymerase activity, as well as the proportion of alpha-amantin-sensitive and resistant during activity, have been measured in the posterior silk glands of the silkworm as a function of growth the fifth larval instar. During the first 5 days, termed the growth phase, the total enzyme activity and particularly the portion that is alpha-amantin-resistant increases to reach a peak value and thereafter declines during the secretory phase, Much of the enzyme remains firmly bound and insoluble. Heparin only only does not inhibit this insoluble and probably chromatin-bound activity which would indicate lack of initiation, but it enhances the activity. A large proportion of newly transcribed RNA is released from the transcription complex. The synthesis of RNA has been studied both qualitatively and quantitatively during the same period. RNA synthesis becomes important on the second day of the fifth instar, as does the RNA polymerase, and stays at a high level for several more days. The results from these studied as well as those with incorporation of 32P indicate interference of varying precursor pools in quantitatively measured RNA synthesis. However, RNA content as well as RNA synthesis in vitro show a close correlation with RNA polymerase activity. The labeled RNAs extracted at different days of the fifth instar have been fractioned on sucrose gradients; this demonstrated that the predominant product of RNA synthesis, as followed by [3H]uridine incorporation at short time intervals, is 45-S preribosomal RNA and 4-5 S RNA. The 45-S RNA is transformed to 19-S and 30-S ribosomal RNA as time progresses or after a chase with unlabeled and/or actinomycin D. There also exists a component heavier than 45S which is fairly rapidly labeled to a small extent.
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PMID:RNA polysomes and RNA synthesis in the silk glands of the silkworm Bombyx mori;. 83 21

The RNA polymerase of the extremely thermophilic bacterium Thermus aquaticus T 2 has been purified to homogeneity. The apparent molecular weights of the subunits of the enzyme are : 165 000, 130000, 92 000 and 44 000. The in vitro temperature optimum of enzyme activity is around 65 degrees C. The enzyme has a preference towards the homologous template and is strongly inhibited by KCI. Rifampicin inhibits the enzyme only to 50% even at very high concentrations. Heparin inhibits it completely, but only at higher molar excess than in the case of the Escherichia coli enzyme. The enzyme can form heparin-resistant complexes at elevated temperatures on the homologous template, but not on E. coli DNA.
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PMID:Purification and properties of the RNA polymerase of an extremely thermophilic bacterium: Thermus aquaticus T2. 94 94

The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e. non-specific fashion. Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.
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PMID:Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A. 109 73

The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin.
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PMID:Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides. 114 63

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.
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PMID:Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii. 162 Jun 19

A 66 base-pair (bp) DNA template carrying a site-specifically placed psoralen cross-link downstream from a phage T7 promoter was constructed. This template can support transcription by T7 RNA polymerase. Transcription was blocked specifically at the psoralen cross-link. We studied the characteristics of elongation complexes, formed in this manner, by enzymatic and chemical footprinting and by a nitrocellulose filter-binding assay. The DNase I footprint of the elongation complex was quantified on a residue by residue basis. It was found that T7 RNA polymerase made the strongest contacts in the central region of the footprint whereas the leading and lagging edges of the polymerase were weakly bound to the DNA. Reducing the NaCl concentration in the transcription reaction resulted in the visualization of two T7 RNA polymerase molecules bound to the same template. A leading polymerase molecule, arrested at the psoralen cross-link, showed a much smaller DNase I footprint than a lagging polymerase molecule that was bound upstream. This upstream polymerase molecule occupied approximately one-half of the promoter region and therefore did not achieve complete promoter clearance. These experiments suggest that complete promoter clearance is required for a gross conformational change in the polymerase, consisting of a contraction in the size of its footprint to occur. DNase I footprinting also revealed that an elongation complex arrested at a psoralen cross-link undergoes several subtle changes in structure in a time-dependent manner and therefore can be considered to be in a state of dynamic flux. Methylation protection showed that some G residues in the top (non-coding) strand are protected against attack by dimethylsulfate, whereas the G residues on the bottom (coding) strand appear not to be protected from reaction with dimethylsulfate. We probed the transcribing complexes for single-stranded regions with T7 gene 3 endonuclease. From the pattern of sensitivity to T7 gene 3 endonuclease on the template strand, we conclude that the RNA-DNA hybrid in the elongation complex is about 7 bp. A nitrocellulose filter-binding assay showed that the elongation complex, consisting of a 36 (+1) nucleotide RNA, the 66 bp DNA template and the T7 RNA polymerase was stable for at least 30 minutes at high salt concentrations. Heparin caused the quantitative release of 36 (+1) RNA nucleotides within 30 seconds, but the DNA was not simultaneously released from the elongation complex under these conditions.
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PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes. 194 44


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