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)

The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper. DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected. While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove. These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.
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PMID:The effect of the minor groove binding agent DAPI (4,6-diamidino-2-phenyl-indole) on DNA-directed enzymes: an attempt to explain inhibition of plasmid expression in Escherichia coli [corrected]. 216 Mar 98

An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate. A second set of primers (nested set) located within the outer set was used in PCR with a known template to prepare the probe. One primer of the nested set included the T7 RNA polymerase promoter at its 5' end allowing transcription of a single-stranded RNA probe. Ten copies of HIV-1 DNA could be detected by PCR-EIA (42 fluorescent units with a background of 18 fluorescent units) compared with a detection limit of 1000 copies by ethidium bromide-stained agarose gel. HIV-1 DNA was detected by PCR-EIA in peripheral blood mononuclear cells from 32 of 33 seropositive patients (range 54-810 fluorescent units), and 0 of 25 seronegative patients (range 20-40 fluorescent units) (sensitivity 97%; specificity 100%). PCR-EIA offers a practical and nonisotopic method to objectively measure PCR-amplified HIV-1 DNA and has the potential for the measurement of other microbial pathogens in human body fluids.
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PMID:Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA. 174 86

HO-221, N-[4-(5-Bromo-2-pyrimidinyloxy)-3-chlorophenyl]-N'-(2-nitrobenzoyl ) urea is a novel benzoylphenylurea derivative. We previously reported HO-221 showed significant antitumor activities against various experimental tumor models, and was especially effective against the solid tumor. In this report we studied the mechanism of action of the compound. The inhibitory activity of HO-221 and 6 kinds of antitumor agents on DNA polymerase alpha was examined in vitro. HO-221 inhibited DNA polymerase alpha activity strongly. From the comparison with IC50 values of individual agents, the inhibitory activity of HO-221 was almost equivalent to aphidicolin and ara-CTP. By double reciprocal plot analysis, the inhibition of HO-221 was found to be non-competitive with the dCTP unlike that of aphidicolin and ara-CTP. Furthermore, HO-221 showed almost no effect on RNA polymerase activity and the protein synthesis. The effect of HO-221 on cell cycle progression of HL-60 cells was examined by flow cytometry analysis. The compound accumulated cells at S phase at a low concentration. The compound showed accumulation of cells in G1, G1-S and G2 + M phases. At higher concentrations, HO-221 increased the G1 phase of tumor cells, stopping the cell cycle progression. Therefore, G1 and S phase accumulation by HO-221 was considered to be correlated with the inhibition of DNA polymerase alpha dependent DNA synthesis. These results suggest that HO-221 is a novel antitumor agent with different mechanism of action from the known antitumor agents.
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PMID:[Mechanism of antitumor effect of a benzoylphenylurea derivative, HO-221]. 226 Aug 70

We introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and DNA. Changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to DNA segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and DNA. We have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific DNA-binding proteins involved in transcriptional regulation (the cro repressor of coliphage lambda, the lac repressor of Escherichia coli, and the RNA polymerase of coliphage T7) and their target DNA fragments ranging in length from 17 to 36 base pairs. The technique allows one to follow complex formation while varying solution conditions such as temperature, pH, ionic strength, and presence of effector molecules. Macromolecular concentrations ranging from 10(-7) to 10(-4) M can be used, allowing estimates of relative binding affinities. The magnitude of the observed rotational correlation times (phi obs) can be used to infer information about the size and shape of the complexes.
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PMID:Detection of protein-DNA complex formation by time-resolved fluorescence depolarization of bound ethidium bromide. 229 77

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.
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PMID:Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels. 235 55

Protein extracts from the protozoan ciliate Paramecium tetraurelia revealed high levels of RNA-dependent DNA polymerase activity (reverse transcriptase). Stable and constant during the somatic phase of the cell cycle, the reverse transcriptase activity quickly diminished following the completion of the sexual phases of the cell cycle: conjugation and autogamy. The Paramecium reverse transcriptase presented a number of common features with retroviral polymerases: ability to copy synthetic templates such as poly(rCm).oligo(dG) as well as mRNA; sensitivity to various reverse transcriptase inhibitors such as HPA 23, suramin, phosphonoformate and ethidium bromide; insensitivity to the action of other DNA and RNA polymerase inhibitors and, finally, the requirement for divalent cations before the enzyme can function: either magnesium or manganese. Although the reverse transcriptase activity was not proven to be independent from one of the DNA polymerases in paramecia, its high activity predicts a role in the paramecia cell cycle. From what we are able to conceive today two possible roles could be envisaged. Participation in the anlage macronucleus formation: micronuclear sequences are first transcripted and, after rearrangements of the RNA molecules, these are retrotranscribed into the macronuclear DNA molecules or association with retrotransposons that participate in the movement of certain macronuclear sequences into the germ-line micronucleus.
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PMID:RNA-dependent DNA polymerase activity in Paramecium tetraurelia: what for? 243 48

A set of four RNA hairpin helices has been prepared by in vitro transcription with T7 RNA polymerase. The hairpins all contain the same nine base pair helix, but with an extra A, C, or U residue forming a bulge at one position; the fourth hairpin is a perfect helix with no bulge. The helix with a bulged A duplicates six base pairs of a helix in the 16S rRNA known to have an unusually high affinity for ethidium bromide [J. M. Kean, S. A. White, and D. E. Draper, Biochemistry 24, 5062 (1985)]. Binding and chemical cleavage studies with ethidium or the reagent methidiumpropylEDTA-Fe(II) [MPE-Fe(II)] showed that the sequence CpG is a preferred intercalation site whether or not a bulge is present; all three bulged bases enhance intercalation at the CpG sequence by an order of magnitude; and intercalation in a bulged helix results in a concerted conformational change involving the entire helix backbone, again dependent on the presence of a bulge but independent of the particular base. These results suggest that an extra sugar-phosphate residue in an RNA helix backbone has a dramatic effect on the ability of the RNA to adopt new conformations. This effect could be an important reason for the conservation of bulges at certain positions in ribosomal and other RNAs.
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PMID:Single base bulges in small RNA hairpins enhance ethidium binding and promote an allosteric transition. 243 51

Transcription of synthetic DNA by T7 RNA polymerase was used to obtain oligoribonucleotides of defined sequence. The enzyme's ability to transcribe DNA immobilized on hydrazide-sepharose was revealed. DNA templates used in such synthesis can be constructed by means of enzymatic (DNA ligase) or chemical ligation (cyanogen bromide).
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PMID:[RNA synthesis using T7 phage RNA polymerase: transcription of synthetic DNA templates in solution and on polymer support]. 247 95

The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.
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PMID:The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth. 247 83

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.
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PMID:Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling. 249 79


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