Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.
Saxton, Matthew N
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Forero-Quintero, L. S., Raymond, W., Handa, T., Saxton, M. N., Morisaki, T., Kimura, H., Bertrand, E., et al. (2021). Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.. Nature communications, 12 (1)https://doi.org/10.1038/s41467-021-23417-0
The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.
Hela Cells, Humans, RNA Polymerase II, Serine, Green Fluorescent Proteins, RNA, Messenger, Microscopy, Fluorescence, Transcription, Genetic, Phosphorylation, Genes, Reporter, Promoter Regions, Genetic, Time-Lapse Imaging, Spatio-Temporal Analysis, Intravital Microscopy, Single Molecule Imaging
NIGMS NIH HHS (R35 GM124747, R35 GM119728)
External DOI: https://doi.org/10.1038/s41467-021-23417-0
This record's URL: https://www.repository.cam.ac.uk/handle/1810/324472
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/