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)

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

A fluorescence spectroscopy method is described for studying association of RNA polymerase with DNA templates. Using double beam differential fluorescence at excitation and emission wavelengths of 285 and 335 nm, respectively, the new technique discriminates non-specific decrease of fluorescence intensity by addition of DNA from quenching of polymerase fluorescence by protein-nucleic acid interactions. Comparing the results with studies of UMP incorporation into RNA, the Ks-values of template-binding were in good agreement with the values for RNA synthesis, pointing to specific interaction of polymerase and the DNA template measured by the fluorescence method. While E. coli enzyme showed higher affinity for templates measured by the fluorescence method. While E. coli enzyme showed higher affinity for templates such as heat denatured poly [d(A-T)] parsley RNA polymerase I accepted such templates with the interaction of both enzymes with single-stranded DNA-templates. UMP incorporation studies suggest that transcription is a cooperative process.
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PMID:Differential fluorescence and kinetic studies on the template-binding of RNA polymerase from parsley and Escherichia coli. 703 65

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.
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PMID:Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis. 711 96

The catalytic center of wheat germ DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-[(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to [3H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The Ki values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 muM respectively. The drugs were covalently bound to the catalytic center by NaBH4 reduction. Both were found bound to the largest enzyme subunit, IIa. It is tentatively concluded that MMPR-OP and AMPR-OP inhibit RNA polymerase II by binding to an essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.
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PMID:Probes of eukaryotic DNA-dependent RNA polymerase II-II. Covalent binding of two purine nucleoside dialdehydes to the initiation subsite. 713 56

Purified ornithine decarboxylase (EC 4.1.1.17, ODC) transamidated with four putrescine moieties on four glutamine residues through the action of transglutaminase (EC 2.3.2.13, TGase) purified from guinea pig liver, when added to isolated rat liver nuclei, stoichiometrically increased the activity of RNA polymerase I (EC 2.7.7.6). The increase was relative to the pmoles of purified conjugated ODC added to the reaction and could be reinitiated after the reaction had plateaued by the further addition of ODC-putrescine conjugate. The kinetics of the reaction suggest that the ODC-putrescine conjugate was not reused but degraded after each initiation. Otherwise, the rapid plateau would not be observed. The repeated addition of 278 pmoles of purified ODC-putrescine conjugate to rat liver nuclear preparations containing 200 micrograms total protein consistently stimulated the incorporation of 600-700 pmoles UMP/mg protein. We suggest that ODC transamidated by its product putrescine may be the posttranslationally modified 65,000 Mr protein which has been reported by several laboratories to serve as a labile subunit of RNA polymerase I.
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PMID:Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity. 715 Mar 60

The ATP analog 5'-adenylyl imidodiphosphate (AMP-PNP) inhibits transcription of specific genes by the RNA polymerase II contained in whole cell extracts, not only with promoters that contain A as the first nucleotide of the transcript, but also with those that initiate transcripts with G or U. The analog AMP-PNP (a competitive inhibitor of ATP) probably acts at the level of initiation of transcription, but it can be used for elongation by RNA polymerase II in isolated nuclei or in the whole cell extract. AMP-PNP and the other imidotriphosphates have little effect on purified HeLa cell RNA polymerase II initiation and elongation of transcription. Since RNA polymerase III in the crude system both initiates and elongates transcripts with AMP-PNP, we conclude that the availability of the beta-gamma bond of ATP is an indispensable requirement for faithful and specific in vitro initiation only by RNA polymerase II in the whole cell extract. Uncapped U- or G-initiated transcripts were obtained in the presence of UMP-PNP or GMP-PNP, the respective imidodiphosphate analogs. The presence of the 5'-terminal imidotriphosphate at the same oligonucleotide as the cap for U-initiated precursors established that transcription initiation and capping occur at the same site. Capping is not required for transcription by RNA polymerase II in the in vitro system. Methylation of the 2' ribose of the initiating nucleotide does not occur on the imidonucleotide containing 5' ends of adenovirus EIV or murine leukemia virus long terminal repeat.
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PMID:Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts. 715 Nov 73

The effect of three components of the anthracycline antibiotic violamycin on the transcription of bacteriophage T3 DNA by bacteriophage T3-induced RNA polymerase has been investigated in a cell-free system. The glycosides of violamycin BI possess the highest inhibitory activity, whereas those of violamycin BII and violamycin A show a reduced inhibitory effect. Concentrations of violamycin BI depressing the incorporation of (3H)UMP into RNA chains have only a slight effect on the binding of the T3 RNA polymerase to T3 DNA and on the incorporation of GTP as the first nucleotide. This shows that the primary target of the antibiotic is not the initiation of the RNA synthesis. The binding of violamycin BI to T3 DNA causes a strong reduction of the elongation rate of the RNA chains.
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PMID:On the mechanism of inhibitory effect of violamycin antibiotics on the transcription by bacteriophage T3-induced RNA polymerase. 723 4

DNA dependent RNA polymerase II was purified to approximately 8300 fold from sonicated nuclear extract of cherry salmon (Onchorhynchus masou) liver by the following purification steps: polyethylene glycol treatment, DEAE=Sephadex A-25 column chromatography, heparin-Sepharose column chromatography, and affinity chromatography on DNA-cellulose. Final preparation of this enzyme has a specific activity of 157 nmole UMP incorporation into RNA per mg of protein per 10 min at 25 degrees. RNA polymerase I was also purified to approx. 3800 fold in a similar manner. Its specific activity was calculated as 26.2 nmole/mg/10 min. Utilization of various UTPs of these enzymes was studied by substitution experiments under the condition of limited synthesis. 5-Methyl UTP (rTTP) could be utilized by the RNA polymerase I 1.7 fold more efficiently compared with UTP. In contrast, the RNA polymerase II recognized rTTP as a substrate as efficiently as UTP. Similar experiments using other alkyl UTPs have been performed.
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PMID:Utilization of 5-alkyl UTPs by DNA-dependent RNA polymerase I and II purified from cherry salmon (Onchorhynchus masou) liver. 725 91

The results of examination of the template activity of the fixed polytene chromosomes of Drosophila hydei, monitored by 3H-UTP, under in situ assay conditions, upon the use of endogenous Drosophila polymerase, exogenous Escherichia coli RNA polymerase (holoenzyme) and exogenous Drosophila RNA polymerase II (or B) have been presented. Analysis of the data reveals that the transcription patterns with the 3 enzymes are not strictly comparable with the pattern obtained under in vivo conditions. Yet, with each of the 3 conditions of assay, there is a reasonable concordance between the template activity on the single X chromosome of the male and the paired Xs of the female, as observed under in vivo. There is also, in every case, a high positive correlation between the 3H-UMP incorporation into the X chromosome and that into a specific autosome. A site-wise analysis of 3H-UMP labelling under the 3 assay conditions also reveals that for most of the regions, the sites which are highly active in vivo also show high labelling in situ, and the proportionally is maintained in both sexes. These result have been interpreted to have suggested that the hyperactivity of the male X vis-a-vis dosage compensation in Drosophila is primarily a property of the inherent organization of the X chromosome itself and is achieved through modulation in the organization, rather than exclusively through autosomal factor(s), although a secondary level of autosomal regulation has not yet been ruled out.
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PMID:Chromosomal basis of dosage compensation in Drosophila. X. Assessment of hyperactivity of the male X in situ. 726 83

Endogenous RNA polymerase activity of isolated nuclei from Physarum polycephalum was determined at high (400 mM KCl) and low (5--100 mM KCl) ionic strength. The activity of RNA polymerase B (alpha-amanitin-sensitive UMP incorporation) and of RNA polymerase A (plus C) (alpha-amanitin-resistant UMP incorporation) was compared in accurately sized nuclear samples derived from macroplasmodia at distinct points of the mitotic cycle. Minimum total RNA polymerase activity was detected in metaphase nuclei. A constant level of RNA polymerase B activity was detected at all other stages of the mitotic cycle, if nuclei were assayed at high ionic strength. However, a high level in S-phase, a low level in G2-phase and again a high level in early prophase were measured, if nuclei were assayed at low ionic strength. Inhibition of DNA synthesis by hydroxyurea in vivo had a selective and drastic effect on in vitro RNA polymerase activity of isolated nuclei derived from S-phase plasmodia, yielding up to 100% inhibition in early S-phase.
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PMID:More evidence for replication-transcription-coupling in Physarum polycephalum. 736 77


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