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)

When incubated with a soluble extract from large oocytes of Xenopus laevis, recombinant DNA plasmids containing either X. laevis oocyte 5S DNA or X. borealis oocyte 5S DNA direct the synthesis of discrete 5S RNAs, which by size and sequence analysis are similar or identical to the corresponding 5S RNAs synthesized in vivo. Synthesis of the 5S RNAs is mediated by a soluble endogenous RNA polymerase III (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6), which presumably recognizes specific initiation and termination sites in the 5S genes. Optimal conditions for accurate synthesis and the kinetics of the reactions have been determined. A soluble postchromatin supernatant fraction has also been isolated from immature oocytes. Although devoid of a functional endogenous RNA polymerase III, this extract contains a component(s) that effects the accurate transcription of 5S genes (in a plasmid) by a purified RNA polymerase III.
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PMID:Transcription of cloned Xenopus 5S RNA genes by X. laevis RNA polymerase III in reconstituted systems. 28 25

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

Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 x 10(4) molecules of RNA polymerases I and III (altogether) and 1 x 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 x 10(4)-2.5 x 10(4) form-I and -III enzyme molecules (altogether) and a further 7 x 10(3)-8 x 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.
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PMID:A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei. 53 87

E. coli fMet-tRNAfMEet and E. coli RNA plymerase (RNA nucleotidyltransferase; EC 2.7.7.6; nucleoside-triphosphate:RNA nucleotidyltransferase) form a 1:1 complex with an apparent association constant of 9.0 X 10(6)M-1 at 37 degrees. The affinity of polymerase to tRNA depends on the tRNA as well as the formyl methionine moiety. Core polymerase has a greatly reduced affinity for initiator tRNA. Optimal binding conditions are similar to those that are also optimal for binding initiator tRNA to ribosomes. Binding of initiator tRNA to polymerase stimulates the transcription of lambda plac DNA, as determined in a crude cell-free system for beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) synthesis as well as in a highly purified transcription system.
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PMID:Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase. 78 83

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.
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PMID:Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. 132 Dec 76

We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will accurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100-200 mM KCl, 12 mM MgCl2), template concentration (20-40 micrograms/ml), incubation time (30-60 min), and temperature (25 degrees C) for class II gene transcription are described.
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PMID:In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells. 141 31

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.
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PMID:An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts. 153 Dec 71

The transcription from the spoVG promoter of Bacillus subtilis is induced at the start of the stationary phase of growth and is dependent on the expression of the spoOA, spoOB, and spoOH genes. It is repressed in cells grown in the presence of excess glucose and glutamine and is under the negative control of the abrB gene. The spoOA and spoOB gene products function to suppress the negative control exerted by abrB. Transcription initiation requires the form of RNA polymerase holoenzyme that contains the spoOH gene product, sigma H. Optimal transcription also requires an upstream A-T-rich region termed the upstream activating sequence (UAS). The mechanism of UAS function was examined through mutational analysis of the spoVG promoter region. Deletion of the UAS or positioning the UAS one half turn or one full turn of the DNA helix upstream of its location in wild-type spoVG resulted in a severe reduction in promoter activity. Deletion of most of the UAS abolished the abrB-dependent repression of spoVG transcription. Higher activity was observed when the UAS was inserted 10 bp (one turn of the helix) upstream than when the sequence was repositioned either 5 or 13 bp upstream. Sequences upstream of the UAS were found not to be involved with the position-dependent function of the UAS. Positioning the UAS 42 or 116 bp upstream eliminated the stimulatory effect of the sequence on spoVG transcription. These data indicate that the UAS functions effectively when it is in close proximity to the -35 region. In vitro transcription analysis indicated that the deletion and insertion mutation affecting the UAS impair RNA polymerase-spoVG promoter interaction. Deletion of the UAS showed that the negative effect of exogenous glucose and glutamine is not dependent on the UAS but is exerted at a site within or near the -35 and -10 regions.
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PMID:Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis. 193 51

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).
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PMID:Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii. 212 Jan 86

Initiated transcription complexes were exposed to adriamycin for up to 48 h. Subsequent elongation of the transcription complex revealed the presence of a series of discrete long-lived blockage sites. The mole fraction of blocked transcripts increased linearly with reaction time, adriamycin concentration, and Fe(III) concentration. Optimal conditions for formation of the blocked transcript were 24-h reaction time, 10 microM adriamycin, and 75 microM Fe(III) ions. Nine high-intensity blocked transcripts were observed, and all correspond to transcription proceeding up to G of GpC sequences of the nontemplate strand. The presence of 75 microM Fe(III) ions enhanced the amount of transcriptional blockages by 12-15-fold. Two blocked transcripts decayed with a half-life of 0.32 and 1.9 h, and one of these exhibited 100% effective delayed termination 6 bp downstream of the original blockage site. All other blockages were unchanged after 3 h of elongation. Bidirectional transcription footprinting was used to define the physical size of the drug-induced blocking moiety as a maximum of 2 bp, and this was observed at all three GpC elements probed by RNA polymerase from both directions. The nature of the apparent covalent adducts has not yet been established but is probably a G-specific adduct deriving from a reduced form of the drug (quinone methide). Although the GpC specificity suggests an interstrand G-drug-G cross-link, these were not detected by heat denaturation and subsequent denaturing gel electrophoresis of the end-labeled promoter fragment.
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PMID:Induction of stable transcriptional blockage sites by adriamycin: GpC specificity of apparent adriamycin-DNA adducts and dependence on iron(III) ions. 238 92


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