Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

Linda S. Forero-Quintero, William Raymond, Tetsuya Handa, Matthew N. Saxton, Tatsuya Morisaki, Hiroshi Kimura, Edouard Bertrand, Brian Munsky () and Timothy J. Stasevich ()
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Linda S. Forero-Quintero: Colorado State University
William Raymond: Colorado State University
Tetsuya Handa: Tokyo Institute of Technology
Matthew N. Saxton: Colorado State University
Tatsuya Morisaki: Colorado State University
Hiroshi Kimura: Tokyo Institute of Technology
Edouard Bertrand: Tokyo Institute of Technology
Brian Munsky: Colorado State University
Timothy J. Stasevich: Colorado State University

Nature Communications, 2021, vol. 12, issue 1, 1-16

Abstract: Abstract 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.

Date: 2021
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DOI: 10.1038/s41467-021-23417-0

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