Different populations of RNA polymerase II in living mammalian cells

Author： Hieda Miki Winstanley Henry Maini Philip Iborra Francisco Cook Peter

ISSN： 0967-3849

Source： Chromosome Research, Vol.13, Iss.3, 2005-01, pp. : 135-144

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

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-β-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. II_A). 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. II_O).