Effects of Anticancer Drugs on Transcription in vitro

2001 ◽  
Vol 56 (9-10) ◽  
pp. 886-891 ◽  
Author(s):  
Dorota Wilmańska ◽  
Malgorzata Czyz ◽  
Kazimierz Studzian ◽  
Mariola K. Piestrzeniewicz ◽  
Marek Gniazdowski
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Ethidium Bromide ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
T7 Rna Polymerase ◽  
Sequence Specificity ◽  
Total Rna ◽  
Abortive Initiation

AbstractThe effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E.coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E .coli RNA polymerase (“abortive initiation“); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter ϕ 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G · C ) specificity (actinomycin D), the non-intercalating, d(A · T ) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

scholarly journals Effect of DNA-interacting drugs on phage T7 RNA polymerase.

1998 ◽  
Vol 45 (1) ◽  
pp. 127-132 ◽  
Author(s):  
M Piestrzeniewicz ◽  
K Studzian ◽  
D Wilmańska ◽  
G Płucienniczak ◽  
M Gniazdowski
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
T7 Rna Polymerase ◽  
Distamycin A ◽  
E Coli ◽  
Phage T7 ◽  
Drug Dependent ◽  
Dna Complex ◽  
Kinetics Of

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.

scholarly journals RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES

1973 ◽  
Vol 57 (2) ◽  
pp. 538-550 ◽  
Author(s):  
R. Sederoff ◽  
R. Clynes ◽  
M. Poncz ◽  
S. Hachtel
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Polytene Chromosomes ◽  
Polymerase Activity ◽  
Exogenous Rna ◽  
Rna Polymerase Activity ◽  
Chromosome Regions

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.

scholarly journals RNA polymerase activity in bovine spermatozoa.

1977 ◽  
Vol 74 (3) ◽  
pp. 698-706 ◽  
Author(s):  
C D Fuster ◽  
D Farrell ◽  
F A Stern ◽  
N B Hecht
Keyword(s):  
Rna Polymerase ◽  
Sodium Hydroxide ◽  
Ethidium Bromide ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Polymerase Activity ◽  
Intracellular Location ◽  
Bovine Spermatozoa ◽  
Rna Polymerase Activity ◽  
Mature Spermatozoa

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.

scholarly journals Syn5 RNA polymerase synthesizes precise run-off RNA products

2013 ◽  
Vol 42 (5) ◽  
pp. e33-e33 ◽  
Author(s):  
Bin Zhu ◽  
Stanley Tabor ◽  
Charles C. Richardson
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Promoter Sequence ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Intermediate Products ◽  
Run Off ◽  
Rna Fragments ◽  
Single Subunit

Abstract The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3′-terminus (N + 1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30 000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3′-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3′-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

RNA SYNTHESIS IN RAT LIVER AFTER CORTISOL TREATMENT: A POSSIBLE MITOCHONDRIAL-NUCLEAR RELATIONSHIP

1974 ◽  
Vol 77 (2) ◽  
pp. 298-309 ◽  
Author(s):  
A. Maher Mansour ◽  
Sylvan Nass
Keyword(s):  
Rna Polymerase ◽  
Ethidium Bromide ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Ovariectomized Rats ◽  
Saline Control ◽  
Mitochondrial Rna ◽  
Nuclear Rna Synthesis ◽  
Hormone Injection ◽  
Nuclear Rna

ABSTRACT Liver mitochondrial RNA synthesis (as measured by an RNA polymerase assay) in adrenalectomized ovariectomized rats increased (42 %) 1 h after injection of 0.5 mg cortisol/100 g b. wt. Nuclear RNA synthesis increased (130–171 %) 4 h after injection of 4 mg cortisol/100 g but did not increase after treatment with the lower dosage of cortisol after 4 h. There was no effect on the nuclei from either 0.5 or 4 mg cortisol after 1 h. If either ethidium bromide, rifampicin or actinomycin D were injected 10 min before the hormone injection, a complete inhibition of the hormonal action on mitochondrial RNA synthesis was found. On the other hand, nuclear RNA synthesis was partly inhibited if either ethidium bromide or rifampicin preceded the hormone injection. Actinomycin D inhibited nuclear RNA polymerase below the saline control level. Assuming that ethidium bromide and rifampicin act specifically on mitochondrial RNA synthesis, it would appear that there is a mitochondrial component to the RNA synthesis measured in the nucleus after cortisol treatment.

scholarly journals Efficient inhibition of RNA self-primed extension by addition of competing 3′-capture DNA-improved RNA synthesis by T7 RNA polymerase

2019 ◽  
Vol 47 (19) ◽  
pp. e118-e118 ◽  
Author(s):  
Yasaman Gholamalipour ◽  
William C Johnson ◽  
Craig T Martin
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
T7 Rna Polymerase ◽  
Current Work ◽  
High Yield ◽  
Rna Biology

Abstract In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3′ end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during ‘high yield’ transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.

CHANGES IN LABELLING OF RIBONUCLEIC ACID SPECIES FRACTIONATED FROM THE RAT VENTRAL PROSTATE IN RESPONSE TO TESTOSTERONE

1975 ◽  
Vol 78 (2) ◽  
pp. 401-416 ◽  
Author(s):  
Anja Isotalo ◽  
R. S. Santti
Keyword(s):  
Body Weight ◽  
Glucose Metabolism ◽  
Ribosomal Rna ◽  
Orotic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Ventral Prostate ◽  
Total Rna ◽  
Testosterone Administration

ABSTRACT The total RNA isolated at various times up to 24 h after testosterone administration from the ventral prostate of castrated rats, was labelled either by injecting 3H-orotic acid directly into the ventral prostate 6 h before the animals were killed, or by incubating prostatic tissue in vitro with 3H-uridine for 20 to 60 min. The isolated RNA was separated into tRNA, ribosomal RNA (Q1 RNA) and two DNA-like RNA fractions (Q2) and TD RNA) by chromatography on methylated albumin kieselguhr (MAK) columns, and the fractions were further analysed by sucrose gradient centrifugation. Testosterone given into castrated animals for 12 h, stimulated the labelling of all main fractions. The radioactivity of TD RNA after a 60-min incubation period in vitro with 3H-uridine was approximately twice that seen in the castrated rat, while there was a 3.1- and 2.2-fold increase in the radioactivity of the Q1 and Q2 RNA fractions respectively. Kinetics of incorporation of 3H-uridine into different RNA fractions revealed that the hormone facilitated the labelling of the TD RNA fraction relatively more than that of the Q2 fraction. The injection of 3H-orotic acid into the ventral prostate labelled the Q1 RNA preferentially. More than 60 % of the recovered radioactivity was found in Q1 RNA (as 18 and 28 S). Testosterone increased markedly (9.4-fold) the labelling of this fraction. It was concluded that testosterone has an activatory effect on the production of ribosomal RNA, and the bulk of the testosterone effect on the total RNA labelling is to be found in this fraction. Furthermore, it seems likely that testosterone also stimulates both the synthesis and processing of DNA-like RNA. When actinomycin D was given 2 h before the hormone administration in a dose of 25 μg per 100 g of body weight, there was no noticeable increase in the labelling of any fraction above the level seen in the untreated castrated rat. There is evidence that testosterone exerts some effects on the labelling of proteins with radioactive amino acids and 14C-glucose metabolism in the absence of that fraction of the total RNA synthesis which is sensitive to a low dose (25 μg per 100 g of body weight) of actinomycin D (Isotalo & Santti 1972). In this way it may be concluded that the major changes of the RNA synthesis after testosterone administration are likely to be secondary to the protein synthesis and glucose metabolism, or the hormone exerts its anabolic effect on prostatic cells at different sites and by different modes of action, each of which can be operated independently.

scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

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