Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells.
RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors--some of which are used therapeutically--affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.
Fromaget
Maud
M
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.
Cook
Peter R
PR
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000378:metabolism; D000818:Animals; D001552:Benzazepines / Q000378:metabolism; D002460:Cell Line; D002945:Cisplatin / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000378:metabolism; D003609:Dactinomycin / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005260:Female; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D038761:Photobleaching; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D001552:Benzazepines; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D007211:Indoles; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; C120793:alsterpaullone; D003609:Dactinomycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D012319:RNA Polymerase II; D046988:Proteasome Endopeptidase Complex; D006655:Histone Deacetylases; D002945:Cisplatin