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Published 9 December 2002. doi:10.1083/jcb.200206019
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© The Rockefeller University Press, 0021-9525/2002/12/777 $5.00
The Journal of Cell Biology, Volume 159, Number 5, 777-782

Article

 The transcription cycle of RNA polymerase II in living cells

Hiroshi Kimura, Kimihiko Sugaya and Peter R. Cook

Sir William Dunn School of Pathology, Oxford OX1 3RE, UK

Address correspondence to Peter R. Cook, Sir William Dunn School of Pathology, South Parks Rd., Oxford OX1 3RE, UK. Tel.: 44-1865-275528. Fax: 44-1865-275515. E-mail: peter.cook{at}path.ox.ac.uk

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: ~75% moved rapidly, but ~25% was transiently immobile (association t1/2 {approx} 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-ß-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 {approx} 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.

Key Words: green fluorescent protein; photobleaching; actinomycin D; DRB; kinetics


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