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: ∼75% moved rapidly, but ∼25% was transiently immobile (association t1/2 ≈ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-β-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 ≈ 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.

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…As for FRAP, after FVP treatment we detected the dramatic dissociation of Pol II from chromatin (Fig EV3A), in agreement with previous observations (Kimura et al , 2002; Hieda et al , 2005). However, both in the case of the highly mobile PIC components, TAF5 and TFIIB (Fig EV3B and C) and the tested SAGA and ATAC subunits (Fig EV3D–H), FLIP analyses detected no, or only minor deviations, from the average curves generated from non‐treated cells.…”

“…The FRAP ROI of the GCN5‐expressing cell reached full recovery after the first 10 s (Fig 1A), whereas raw fluorescence intensity in the ROI of the RPB1‐expressing cell did not (Fig EV1A). FRAP kinetics of eGFP, RPB1, TAF5 and TFIIB were in good agreement with previously published data (Kimura et al , 2002; Hieda et al , 2005; de Graaf et al , 2010; Fig EV1B–D).…”

“…Our FRAP and FLIP analyses classified the two coactivators as highly mobile factors, with no detectable immobile fractions, suggesting transient chromatin associations for these complexes. This behaviour is very similar to the dynamic GTFs, TFIID and TFIIB, and distinct from Pol II, which has a significant immobile fraction forming stable interactions with chromatin (Kimura et al , 2002; Hieda et al , 2005). Consequently, we conclude that the recruitment and function of the complexes on their chromatin substrate do not depend on direct and stable interactions with transcribing Pol II.…”

“…First, we chose RPB1 (the largest subunit of Pol II) known to have stable interactions with chromatin (Kimura et al , 2002; Hieda et al , 2005; Boireau et al , 2007; Darzacq et al , 2007; Fromaget & Cook, 2007) and also highly dynamic nuclear factors, like the TFIID subunit TAF5 and the GTF TFIIB (Chen et al , 2002; Sprouse et al , 2008; de Graaf et al , 2010; Ihalainen et al , 2012). In addition, eGFP was used as a control for a freely diffusing protein that should exhibit no specific interactions with the nuclear environment.…”

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

“…In fact, this modulation is one of the key mechanisms essential for the functional role of nuclear hormone receptors (Stenoien et al, 2001;Schaaf and Cidlowski, 2003;Stavreva et al, 2004;Elbi et al, 2004b;Farla et al, 2005;Rayasam et al, 2005). In vivo FRAP has been used to study the dynamic properties of chromatin proteins and that the FRAP recovery kinetics of chromatin proteins are directly related to their chromatin-binding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005). Because BRG1 and BRM were selectively recruited to the MMTV array in response to hormone (Figures 2 and 3), we used FRAP to study the binding kinetics of chromatin-remodeling complexes at the amplified MMTV target in vivo.…”