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 cells are lysed in solutions containing high concentrations of salt and a non-ionic detergent, structures are released which retain many of the morphological features of nuclei. These nucleoids contain superhelical DNA but are depleted of nuclear protein. We have analysed DNA conformation in nucleoids derived from HeLa cells synchronized at different stages in the cell cycle. The gross differences in nuclear morphology seen during the cell cycle are reflected in the morphology of the nucleoids; for example, the individual chromosomes of mitotic cells remain identifiable and aggregated within the mitotic nucleoid. The sedimentation rate of nucleoids in sucrose gradients reflects the gross nuclear morphology; the small S-phase nucleoids sediment 9 times faster than the large mitotic nucleoids. Despite these large differences at the gross level of organization, both the degree of supercoiling and the size of the units in which supercoiling is maintained are roughly similar in the nucleoids derived from cells in the different phases. The protein content of the various nucleoids is also very similar. Like the nucleoids made from randomly growing cultures of cells, mitotic nucleoids are excellent templates for the RNA polymerase of Escherichia coli.
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PMID:Supercoiling of DNA and nuclear conformation during the cell-cycle. 64 87

Hg-UMP-containing transcripts made from chick erythroid chromatins with E. coli RNA polymerase hybridize to chick globin cDNA. Contamination with endogenous globin RNA has been largely removed by purification on SH-agarose columns at 55 degrees C. Some endogenous globin mRNA sequences remain, probably as hybrids with "anti-sense" Hg-transcripts produced by RNA-dependent RNA synthesis. Heating to 115 degrees C before SH-agarose chromatography eliminates these contaminants. Hg-transcripts from adult and embryonic erythroid chromatins purified by this method are hybridized to globin cDNA; they contain a 4- to 6-fold higher proportion of globin-specific sequences (10-13 PPM) than do transcripts from brain chromatin. Dissociation of erythroid chromatins in salt and urea, followed by reconstitution using standard methods, destroys even this low degree of specificity.
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PMID:Is there specific transcription from isolated chromatin? 65 20

Transcription by endogenous RNA polymerase B in lysates of Ehrlich ascites cells was investigated. The enzyme exhibits two salt optima at 0.025 M and at 0.3 M (NH4)2SO4 respectively. Preincubation of the cells with the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole results in an inactivation of the polymerase molecules active under condition of low salt. This indicates two functional states of the enzyme in vivo. Initiations of RNA chains by polymerase B do not occur in vitro as judged by the incorporation of [beta-32P]GTP. Thus the two functional states seem to be both elongating polymerase molecules. Polymerase B does not occur in the lysates in a state ready to initiate on an exogenous template, in contrast to polymerase A and C which do occur in free form. Pretreatment with dichlororibofuranosylbenzimidazole in vivo does not result in an accumulation of free polymerase B.
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PMID:On the activity of RNA polymerase B in lysates from Ehrlich ascites cells. 65 42

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.
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PMID:Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor. 69 29

Crude nuclei were isolated from trunks of 13-day-old chicken embryos under conditions which prevent leakage of RNA polymerases from nuclei. RNA polymerases were solubilized by subsequent incubation in alkaline buffer and sonication at high salt concentration. Purification of RNA polymerases A, B, and C was achieved by conventional column chromatographic procedures. RNA polymerase B was freed from an UTP:polynucleotidyl exotransferase by chromatography on a tRNA-Sepharose column. Purified RNA polymerase A contained six putative subunits with molecular weights 190 000 (A1), 117 000 (A2), 57 000 (A3), 50 000 (A4), 25 000 (A5), 19 000 (A6); RNA polymerase B contained eight putative subunits with molecular weights 98 000 (B2'), 86 000 (B2''), 155 000 (B3), 44 000 (B4), 31 000 (B5), 28 000 (B6), 26 000 (B7), 19 000 (B8); RNA polymerase C contained nine putative subunits with molecular weights 170 000 (C1), 117 000 (C2), 84 000 (C3), 60 000 (C4), 49 000 (C5), 36 000 (C6), 33 000 (C7), 22 000 (C8), 19 000 (C9).
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PMID:Purification of class A, B, and C DNA-dependent RNA polymerases from chicken embryos. 71 15

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 X 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.
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PMID:The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro. 74 48

Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit. The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation.
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PMID:RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant. 76 57

RNA transcribed in vitro at low ionic strength, from either rat liver chromatin or DNA, contains a significant amount of structure resistant to RNase in high salt buffer. This is observed with rat liver (form B polymerase) as well as with Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6). Treatment with RNases specific for either double-stranded or hybrid RNA indicates that resistance to RNase is due to the presence of double-stranded RNA sequences. Denaturation kinetics in the presence or absence of RNase suggest that these sequences are formed by intramolecular base pairing. Their mean length is about 20 to 30 nucleotides, but 15-20% are more than 100 nucleotides long. They contain 60-65% G-C base pairs. The proportion of double-stranded segments is higher in chromatin transcripts than in DNA-templated RNA, and is higher with homologous RNA polymerase than with the bacterial enzyme. On the other hand, chromatin endogenous RNA polymerase, which is unable to initiate transcription, does not synthesize double-stranded RNA. The problem of the location of these sequences is discussed; preliminary results suggest that the 5' end of the RNA transcripts could be enriched in complementary sequences.
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PMID:Double-stranded RNA in chromatin transcripts formed by exogenous RNA polymerase. 77 79

sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.
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PMID:Spatial relationship of the sigma subunit and the rifampicin binding site in RNA polymerase of Escherichia coli. 77 17

Using an assay specific for chain elongation of E. coli RNA polymerase the kinetics of this propagation reaction have been studied. The kinetic behaviour is consistent woth the mathematical model formulated for this multisubstrate enzyme. The effect of increasing salt concentration on the kinetics of the reaction indicated that DNA unwinding is probably a necessary step in the propagation step, although this may not be the rate limiting step under all conditions.
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PMID:The kinetics of E. coli RNA polymerase. 78 32


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