Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.
Linda S Forero-QuinteroWilliam RaymondTetsuya HandaMatthew N SaxtonTatsuya MorisakiHitoshi KurumizakaEdouard BertrandBrian MunskyTimothy J StasevichPublished in: Nature communications (2021)
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.
Keyphrases
- living cells
- single molecule
- fluorescent probe
- protein kinase
- transcription factor
- genome wide
- copy number
- atomic force microscopy
- dna methylation
- induced apoptosis
- genome wide identification
- gene expression
- cell cycle arrest
- high resolution
- human immunodeficiency virus
- cell death
- hepatitis c virus
- high throughput
- single cell
- high frequency
- mass spectrometry
- hiv testing
- genome wide analysis
- small molecule
- binding protein
- label free
- crispr cas
- signaling pathway
- high throughput sequencing
- optical coherence tomography