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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)

The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy. For electron-microscopic investigations the effect of different conditions of formaldehyde fixation for DNA-RNA-polymerase complexes was studied and optimal fixation conditions were found. The suggested fixation method for DNA-RNA-polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ration of enzyme to DNA etc.). The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence. The electron-microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA. A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found.
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PMID:Investigation of the binding of Escherichia coli RNA polymerase to DNA from bacteriophages T2 and T7 by kinetic formaldehyde method and electron microscopy. 33 47

A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected. This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20 degrees C. At temperatures above 30 degrees C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.
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PMID:The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method. 77 Jan 76

Chick liver nuclear 5.0 S RNA, which stimulates RNA synthesis on chromatin, binds preferentially to the deoxyribonucleoprotein in homologous chromatin. The proteins found in the isolated deoxyribonucleoprotein-5.0 S RNA complex are total amount of both H4 and H3 histone, about 20% of nonhistone protein and about 50% of both H2a and H2b histone found in the original chromatin. Cesium chloride equilibrium centrifugation of the deoxyribonucleoprotein-5.0 S RNA complex after fixation with formaldehyde shows that the 5.0 S RNA is bound to certain proteins (acid-soluble and -insoluble) in the complex. The stimulation of RNA synthesis by the nuclear RNA fraction is due to the conversion of inaccessible region of DNA to RNA polymerase into an accessible one and presumably not due to an increase in the number of binding sites for RNA polymerase in the chromatin. The release of non-histone protein from chromatin following the addition of the nuclear RNA fraction is also briefly discussed.
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PMID:Interaction of chromatin components with nuclear 5.0 S RNA fraction that stimulates RNA synthesis. 92 98

The mechanism of the effect of an RNA polymerase II (RNA nucleotidyltransferase II) stimulation factor isolated from the nuclei of chicken myeloblastosis cells was studied. The stimulation requires the presence of all four nucleoside triphosphates and depends upon an exogenous DNA template. In the absence of the factor, RNA synthesis ceases after 20-30 min, but in the presence of the factor, synthesis continues up to 60-80 min. Addition of the factor at 35 min after incubation causes resumption of RNA synthesis. The factor greatly stimulates the activity of RNA polymerase II at low enzyme concentrations. The RNA polymerase activity is more sensitive to alpha-amanitin inhibition when the factor is present. Experiments of [gamma-32P]ATP incorporation reveal that the factor provides for an increased frequency of initiation of RNA chains, both of the primary initiation events and re-initiation after previous ones were completed. A slightly higher rate of RNA chain growth was also observed with this factor but the ultimate size of RNA synthesized was not affected, as determined by formaldehyde/sucrose gradient centrifugation. These data suggest that the factor functions at the initiation stages of the RNA polymerase reaction.
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PMID:Increased frequency of initiation of RNA synthesis due to a protein factor from chicken myeloblastosis nuclei. 105 84

The reality of the nonrandom distribution of histones along the chromatin strand was investigated in several ways. It does not appear to derive from histone exchange during shearing and is evident in chromatin fixed with formaldehyde prior to shearing. Endogenous or Escherichia coli polymerase are preferentially associated with regions of chromatin with a low protein/DNAratio. Although RNA polymerase and histones are fixed to chromatin after formaldehyde treatment with high efficiency, only a minor fraction of non-histone protein is fixed under similar conditions. Even after washing in high salt to minimize adventitious association, most remaining non-histone protein fails to be fixed. The utility of this approach for defining chromosomal proteins is discussed.
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PMID:The nature of protein association with chromatin. 109 33

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
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PMID:Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand. 228 Jul 70

RNA-polymerase of E. coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase. After fixation of the complex with formaldehyde it was analysed by electron microscopy. A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs. The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure. In pBR322 the strong binding sites correlate with the position of promoters. Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content. That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.
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PMID:[Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy]. 266 82

Proteins cross-linked to pBR322 mRNAs and DNA by formaldehyde treatment of intact Escherichia coli cells have been detected with the use of a novel detection method. Among the proteins cross-linked to pBR322 mRNAs were S1, S21, and at least six other proteins of the small ribosomal subunit, initiation factor 1, elongation factor (EF) Tu, and very small amounts of EF-G and EF-Ts. The single strand binding protein, the HU-proteins, and RNA polymerase subunits alpha and beta were among the proteins cross-linked to pBR322 DNA. The results obtained suggest that the procedures described, can also be used to study interactions between different nucleic acid-bound polypeptides. The results are discussed in relation to the working mechanism of formaldehyde, and are compared to the results obtained with cross-linking induced by ultraviolet light. The methods presented should also be of use for the study of nucleic acid-protein interactions in other organisms.
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PMID:Hybridization selection of covalent nucleic acid-protein complexes. 2. Cross-linking of proteins to specific Escherichia coli mRNAs and DNA sequences by formaldehyde treatment of intact cells. 389 62

Contact-site cross-linking agents comprise a heterogeneous grouping of cross-linkers which share the common property of being able to cross-link only very closely juxtaposed residues in macromolecular complexes. We have defined contact-site cross-linking arbitrarily as the covalent joining of residues such that they are constrained to a distance which is equivalent to or less than their closest possible steric approach prior to becoming linked (1). We recognize two classes of contact-site cross-linkers, bridge type and zero-length type. The former, such as formaldehyde, become incorporated during cross-linking as one-atom bridges. The latter, such as the carbodiimides, operate as condensing agents with the result that the cross-linked residues become interjoined directly. Contact-site cross-linkers have been used in several ways as specific probes of both the static and dynamic aspects of macromolecular structure. They can yield precise structural information about macromolecular contacts when actual sites of cross-linking are determined by peptide or nucleotide mapping techniques. In this way exact contacts between histones in the nucleosome, between protein and RNA in the ribosome, and between RNA polymerase and DNA have been determined. Contact-site cross-linkers have also been used to probe the perturbation of contacts following macromolecular conformational changes. Certain histone-histone 'cross-linkable' sites are rendered unreactive after induction of chromatin conformational changes thus serving to localize sites of perturbation.
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PMID:Contact-site cross-linking agents. 611 63

The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis. Methodology was developed to improve their extraction from enriched fractions. Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin. Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II. Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated. Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome. Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication. The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.
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PMID:Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication? 621 Jan 83


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