Colorado State University
Collaborators: William Raymond, Colorado State University; Brian Munsky, Colorado State University; Timothy J. Stasevich, Colorado State University
Title: Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP2) occurs in a spatiotemporal organized manner at a single-copy gene in live-cells
Short Abstract: We visualized, quantified, and modeled the spatiotemporal organization of RNAP2 phosphorylation at a single-copy gene. Using three-color fluorescence microscopy with antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells at single-gene resolution. Applying this methodology in combination with computational models, we found live-cell evidence that genes contain a heterogenous distribution of RNAP2 along their length, with 5-40 copies per transcription site (predominantly phosphorylated in Serine 5 clusters near the promoter). RNAP2 fluctuations are spatially and temporally resolved from those of the nascent mRNA, with most RNAP2 being phosphorylated within 6 seconds, escaping the promoter within ~1.5 minutes, and completing transcription in 5 minutes .
Presenter Biosketch: I am a postdoc in the group of Brian Munsky at CSU. I study transcriptional dynamics in single-copy genes. In the first part of my postdoc, I combined multi-color, single-molecule microscopy with fluorescent antibody-based probes that bind to unphosphorylated and phosphorylated forms of the CTD tail of RNAP2 in living cells. In my present research, I am investigating the gene expression of endogenous pro- and counter-inflammatory genes upon stimulation with Dexamethasone and IL-1B by employing smiFISH and automated image processing. During my Ph.D. research at the University of Kaiserslautern-Germany, I worked on Neurosciences and Brain Energy Metabolism. I characterized the role of monocarboxylate transporters (MCTs) during epileptiform activity using pH and Ca2+ imaging.
Link to Full Abstract: Forero_Linda