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Proc Natl Acad Sci U S A. 2018 May 8;115(19):E4368-E4376. doi: 10.1073/pnas.1717920115. Epub 2018 Apr 9.

Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.

Author information

1
Department of Molecular Genetics, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands.
2
Oncode Institute, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands.
3
Department of Pathology, Optical Imaging Centre, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands.
4
Department of Molecular Genetics, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands; J.Marteijn@erasmusmc.nl.

Abstract

Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell-imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

KEYWORDS:

RNA Polymerase II; live-cell imaging; promoter-proximal pausing; transcription; transcription dynamics

Comment in

PMID:
29632207
PMCID:
PMC5948963
DOI:
10.1073/pnas.1717920115
[Indexed for MEDLINE]
Free PMC Article

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