Live cell analysis of RNA polymerase i transcription. Kinetic modeling of gene regulation and transcriptional control – Pol2Kinetics

RNA polymerase II (pol II) is at the center of cellular phenotypic expression. Pol II transcribes type 2 genes potentiating cellular functions in response to diverse stimulations leading to cellular specialisations defining environmental responses, organs differentiation or even species (Levine and Tjian, 2003). Over the last decades, type 2 transcription has been extensively studied and the enzymatic mechanism of transcription starts to be well understood. It has been possible to reconstitute active enzymes in test tubes allowing to measure in real time the in vitro kinetics of transcription at the single molecule level (Davenport et al., 2000; Margeat et al., 2005). These in vitro studies provide us with the frame of this mechanism. We also possess a multitude of information on transcriptional control by specific transcription factors. What remains largely unclear is how a specific transcription factor can influence the mechanism of transcription to tune its activity specifically on a particular gene. We recently developed methodologies enabling us to measure the specific activity of a particular gene in a live cell (Darzacq et al manuscript in preparation). Here we propose tu put these technologies in application to understand how on different genes the transcriptional machinery is able to perform different tasks. Different transcriptional systems will be investigated initially and later phases of the project will be devoted to the development of higher throughput techniques for studying gene specific kinetics.