Different populations of RNA polymerase II in living mammalian cells.
RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO).
Hieda
Miki
M
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
Winstanley
Henry
H
Maini
Philip
P
Iborra
Francisco J
FJ
Cook
Peter R
PR
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic
D049452:Green Fluorescent Proteins; D004004:Dichlororibofuranosylbenzimidazole; D012319:RNA Polymerase II