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)

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.
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PMID:The inhibition of ribonucleic acid polymerase by acridines. 429 May 34

Subinhibitory doses of rifampin cured F(+)Escherichia coli cells from the episome. The target of the drug was transcription because E. coli mutants with a ribonucleic acid polymerase resistant to rifampin were not cured. The experimental conditions required for optimal curing with rifampin very closely resembled those required for curing with acridine orange. Mutants were found which are more resistant to curing by both acridine orange and rifampin. Probably the two drugs affect a common metabolic step, or alternatively they may inhibit the synthesis of a factor which is necessary for the replication of the episome.
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PMID:Relationships between curing of the F episome by rifampin and by acridine orange in Escherichia coli. 459 25

In vitro transcription of supercoiled DNA by purified E. coli RNA polymerase was inhibited by Acridine Orange in a bimodal manner while N-10 benzyl substituted Acridine Orange is about one-third as inhibitory and effects monophasic inhibition. The inhibition correlates with the supercoil unwinding abilities of these two intercalators with Acridine Orange unwinding supercoiled DNA at 1/3 the concentration required for the substituted acridine orange. Direct visualization of DNA-RNA polymerase complex on agarose gels showed that these intercalators directly interfere with this association and the more effective the drug is in unwinding DNA supercoils the more effective it is in interfering with the DNA-enzyme complex. In addition, specific intercalators differentially affect the stability of DNA-RNA polymerase-RNA ternary complexes.
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PMID:In vitro effects of acridine intercalation on RNA polymerase interactions with supercoiled DNA. 619 30

A number of spontaneous rifampicin-resistant (Rifr) mutants were isolated from a strain of E. coli having a deletion in the lac proA proB region of the chromosome. The stability of a F'lac proA proB episome in these mutants was determined by their sensitivity to acridine orange curing and the frequency of spontaneous loss of episomes. The Rifr mutants can be divided into three classes based on their ability to maintain the F'lac pro episome. Class I mutants (25% of the total Rifr mutants) showed high degree of spontaneous episome loss and high sensitivity to acridine orange curing. Class II mutants (55% of the total Rifr mutants), like the parent strains, showed intermediate sensitivity to acridine orange curing. Class III mutants (21% of the total Rifr mutants) showed high resistance to acridine orange curing and low frequency of spontaneous episome loss. Three-fourths of the Class II mutants were found to be Hfr as shown by their lack of the F'lac pro DNA band on agarose gel together with their ability to mobilize chromosomal markers in mating. Representative Rifr mutants from each class were selected and the Rifr mutants from each class were selected and the Rifr mutations were mapped within the proB gene for the beta beta' operon by P1 transduction. These results indicate that RNA polymerase, or the beta subunit of RNA polymerase, plays an important role in maintaining the F' lac pro episome and in the integration of the F' lac pro episome where no extensive sequence homology is involved.
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PMID:Altered stability and integration frequency of a F' factor in RNA polymerase mutants of Escherichia coli. 702 34

A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays. Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect. Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier. The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction. The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures. This improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.
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PMID:Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA. 875 10

We have developed a new biochemical method to isolate a homogeneous population of RNA polymerase II (RNA pol II) elongation complexes arrested at a DNA damage site. The method involves triple-helix formation at a predetermined site in DNA template with a third strand labeled with psoralen at its 5'-end and a biotin at the 3'-end. After triplex formation and near-ultraviolet irradiation (360 nm), DNA templates modified with psoralen were immobilized on streptavidin-coated magnetic beads and used for in vitro transcription reactions with HeLa nuclear extracts. Separation of magnetic beads from solution results in isolation of arrested elongation complexes on the immobilized DNA templates. We have applied the method to arrest RNA pol II elongation complexes on a DNA template containing HIV-1 promoter. Our results indicate that psoralen crosslink in the template strand efficiently arrests elongation complexes, and psoralen monoadducts terminate transcription. Our results also demonstrate that a triple-helical structure stabilized by an intercalator, acridine, attached to the third strand of the helix inhibits transcription by a termination pathway. Isolation of stable RNA pol II elongation complexes arrested at DNA damage sites is a remarkable finding. This result demonstrates that arrested elongation complexes are impervious to DNA damage repair machinery and other regulatory proteins present in HeLa nuclear extracts. The method of delivering site-specific psoralen damage by a triplex structure and isolation of arrested RNA pol II elongation complexes should be generalizable to any promoter and DNA template sequences. This strategy provides a new approach to study the mechanism of transcription elongation and transcription-coupled DNA damage repair.
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PMID:DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter. 919 26

The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.
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PMID:Inhibition of RNA synthesis in vitro by acridines--relation between structure and activity. 967 27

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.
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PMID:Effect of DNA-interacting drugs on phage T7 RNA polymerase. 970 5

The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase.
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PMID:Crosslinking of cellular DNA by nitracrine and furocoumarin derivatives. 1035 34

The sequence specificity and time course of covalent DNA adduct formation of the novel platinum-acridine conjugate [PtCl(en)(ACRAMTU)](NO(3))(2) [PT-ACRAMTU, 2; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea] have been investigated using restriction enzyme cleavage and transcription footprinting assays and compared to the damage produced by the clinical agent cis-diamminedichloroplatinum(II) (cisplatin, 1). The rate of DNA binding of 1 and 2 was also monitored by atomic emission spectrometry. Restriction enzymes were chosen that cleave the phosphodiester linkage at, or adjacent to, the predicted damage sites. While conjugate 2 selectively protected supercoiled plasmid from cleavage by EcoRI and DraI enzymes at their respective restriction sites, G downward arrow AATTC and TTT downward arrow AAA, 1 inhibited DNA hydrolysis by HindIII and PspOMI at A downward arrow AGCTT and G downward arrow GGCCC (arrows mark cleavage sites) more efficiently. Transcription footprinting using T7 RNA polymerase revealed major single-base damage sites for 2 at adenine in 5'-TA and 5'-GA sequences. In addition, the enzyme is efficiently stalled at guanine bases, primarily in the sequence 5'-CGA where the damaged nucleobase is flanked by two high-affinity intercalation sites of ACRAMTU. While 1 targets poly(G) sequences, the binding of 2 appears to be dominated by the groove and sequence recognition of the intercalator. The biochemical assays used confirm previous structural information extracted from mass spectra of DNA fragments modified by 2 isolated from enzymatic digests [Barry, C. G., et al. (2003) J. Am. Chem. Soc. 125, 9629-9637]. Possible DNA-binding mechanisms and biological consequences of the unprecedented modification of alternating TA sequences by 2, which occurred at a faster rate than binding to G, are discussed.
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PMID:Unique base-step recognition by a platinum-acridinylthiourea conjugate leads to a DNA damage profile complementary to that of the anticancer drug cisplatin. 1522 67


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