Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate

Dreyer, Christine; Hausen, Peter

doi:10.1093/nar/5.9.3325

Cited by 40 publications

(15 citation statements)

References 25 publications

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“…This shows that the residual initiation can still apparently occur with both ATP and GTP as noted previously in Ehrlich ascites cells [13].…”

Section: O F [3ss]atp[s] and 87 Of ["S]gtp[s] Nuclear Incorporation Wsupporting

confidence: 83%

“…This will have to be determined with a well-characterized cucaryotic RNA polymerase assay system. Also, one can not currently identify whether this inhibitor or some metabolically activated form of it [13,18] causes the actual inhibition of transcription. However, the results presented here arongly suggest that inhibition of transcription may be accomplished by 5,6-dichlororibofuranosylbenzimidazole at sevcral levels, one of which appears to be a decreased accumulation of newly initiated transcripts.…”

Section: Discussionmentioning

confidence: 99%

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Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP

Winicov

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1982

European Journal of Biochemistry

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The y-thio analogues of ATP and GTP, ATP [S] and GTP [S], have been used as affinity probes to measure RNA synthesis initiated in vifro in L cell nuclei. 5,6-Dichlororibofuranosylbenzimidazole was found to inhibit total RNA synthesis in vitro and the amount bound by mercury-affinity chromatography. 5,6-Dichloro-l-/L~-ribofuranosylbenzimidazole has been shown to inhibit transcription of eucaryotic heterogeneous nuclear RNA (hnRNA) [l -31 in several cell lines, resulting in almost complete cessation of mRNA appearance in the cytoplasm [3]. Although evidence has been presented that the mechanism of this inhibition of RNA synthesis is by a process of premature termination in late adenovirus transcription 141 and HeLa cells [5], it has been difficult to study its effects on transcription in detail.The L cell nuclear system has been shown to be sensitive to 5,6-dichlororibofuranosylbenzimidazole inhibition of transcription in vitro [6] and might be expected to provide a convenient system in which to investigate the effect on RNA initiated in vitro. The y-thio analogues of purine triphosphates, ATP [S] and GTP [S] have been used previously to measure initiation of RNA in isolated mouse myeloma nuclei [7], Xenopus luevis oocyte nuclei [8] and adenovirus-infected cell nuclei [9]. Resistance of the [y-thioltriphosphates to phosphatases [7,10] allows isolation of the triphosphorylated termini from RNA molecules initiated in vitro by mercury affinity chromatography and even permits characterization of some of the products initiated in vitro [7-91. This paper reports the use of ATP[S] and GTP[S] to detect initiation in mouse L cell nuclei by means of Hg-affinity chromatography and direct incorporation of 3sS-labeled purine 5'-[1;-thio]triphosphate. Accumulation of RNA transcripts initiated in vitro was significantly inhibited by 5,6-dichlororibofuranosylbenzimidazole as measured by both of these methods. MATERIALS AND METHODS Isolation qf Nuclear R N A Labeled in vitroMouse L cell nuclei were prepared as described previously, after pretreatment of cells in vivo with low levels of actino- Boehringer Mannheim supplied the unlabeled [;!-thioltriphosphates; 5,6-dichlororibofuranosylhenzimidazole was a gift from D r A. F. Wagner (Merck, Sharp and Dohme) and was used at 350 pM. This inhibitor concentration was found to give more consistent results between different nuclear preparations used at 1 . 5~ 10' nuclei ml than the 100 pM concentrations used in previous experiments [6].R N A was extracted and separated from mononucleotides on a Sephadex G-50 column as reported [ I l l with the entire polynucleotide peak precipitated by 2.5 vol. ethanol and used in subsequent analyses. RNA Fractionat ionRNA was separated according to s i x class, after extensive denaturation in 80 dimethylsulfoxide ( Me2SO) for 2 min at 60 "C, by sucrose/sodium dodecyl sulfate gradient centrifugation [ll].Mercury affinity chromatography was carried out by adsorbing extracted RNA on an Affigel 50 1 (Bio-Rad) column in the presence of 1 mM EDTA as descr...

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“…This shows that the residual initiation can still apparently occur with both ATP and GTP as noted previously in Ehrlich ascites cells [13].…”

Section: O F [3ss]atp[s] and 87 Of ["S]gtp[s] Nuclear Incorporation Wsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP

Winicov

Button

1982

European Journal of Biochemistry

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“…Ben-zimidazole derivatives are important scaffolds in medicinal chemistry due to their biological and pharmacological activities. These compounds exhibit activity against several viruses include HIV [7] [8], influenza [9], herpes (HSV-1) [10], RNA [11] and human cytomegalovirus (HCMV) [12]. They have also been employed as antihypertensive, antiviral, anticancer, antiulcer, antifungal and untihistamine [13]- [18].…”

Section: Introductionmentioning

confidence: 99%

Green and High Efficient Synthesis of 2-Aryl Benzimidazoles: Reaction of Arylidene Malononitrile and 1,2-Phenylenediamine Derivatives in Water or Solvent-Free Conditions

Habibi¹,

Valizadeh²,

Mollazadeh³

et al. 2015

IJOC

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“…It is not clear whether DRB directly interacts with the RNA polymerase II transcriptional complex to terminate transcription, because DRB does not inhibit hnRNA synthesis in vitro (9,36). Unlike adenosine and some of its analogs, DRB does not have to be phosphorylated to exert its inhibitory effect (10,19), nor does it alter the ribonucleotide precursor pools (20). Thus, the molecular basis for termination of transcription by DRB is not known.…”

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confidence: 99%

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

Funanage¹

1982

Mol. Cell. Biol.

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Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-p-D-ribofuranosylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-Kl. Three independently isolated mutants, DRB6, DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-Kl cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 ,uM, 300 ,uM DRB was required to inhibit 60%o of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRBsensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 ,uM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 ,uM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR_10) in DRB10 and confirmed that the drbR_10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and a-amanitin-resistant cell lines grew in medium containing both DRB and a-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.The components that comprise a functional RNA polymerase II (nucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) transcriptional complex in eucaryotic cells are not well defined. Although purified RNA polymerase II will accurately initiate transcription at the promoters for the conalbumin, ovalbumin, and globin genes in soluble cell-free extracts from cultured mammalian cells (26,40), at least four other components in the soluble extract are necessary for the selective initiation of transcription (27). In addition, purified RNA polymerase II is a structurally complex enzyme composed of at least 10 subunits, the functions of which are largely unknown (33).In procaryotes, mutations affecting RNA polymerase have been used to characterize the subunits of the RNA polymerase holoenzyme and identify regulatory factors associated with the RNA polymerase transcriptional complex (reviewed in 41). Several of the mutations in t Present address:

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Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate

Cited by 40 publications

References 25 publications

Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP

Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP

Green and High Efficient Synthesis of 2-Aryl Benzimidazoles: Reaction of Arylidene Malononitrile and 1,2-Phenylenediamine Derivatives in Water or Solvent-Free Conditions

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

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