title: The transcription cycle of RNA polymerase II in living cells.
abstract: RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: approximately 75% moved rapidly, but approximately 25% was transiently immobile (association t1/2 approximately 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [3H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t1/2 approximately 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases.
authors.affiliation: Sir William Dunn School of Pathology, Oxford OX1 3RE, UK.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Sugaya
authors.forename: Peter R
authors.initials: PR
authors.lastname: Cook
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003609:Dactinomycin / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010507:Periodicity; D038761:Photobleaching; D012319:RNA Polymerase II / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D008164:Luminescent Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D004004:Dichlororibofuranosylbenzimidazole; D012319:RNA Polymerase II