Spatio-termporal analysis of transcription dynamics in vivo

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The most of our knowledge about transcription, its dynamics and regulation was acquired by biochemical and genetic approaches in vitro, or by microscopy experiments after introducing synthetic genes in eucarytotic cells. We will study here transcription dynamics in vivo in a minimally perturbed system using advanced fluorescence microscopy methods, i.e. super resolution, single molecule microscopy and 3-dimensional single‐molecule tracking. We will take advantage of an iconic biological system that has been used for decades to study diverse aspects of the mRNA life cycle: the large Balbiani ring (BR) transcripts within the nuclei of salivary gland cells of Chironomus tentans. In these cells the BR and heat shock transcription sites can visually well be identified. Furthermore, a high number of transcription processes takes place in parallel within the polytene chromosomes, what strongly improves the chance to observe specific molecular events by light microscopy. The goal of this project is to analyze the topology of the transcription sites, the dynamics of transcription and its regulation in vivo under conditions that are as native as possible. We will focus on a number of fundamental questions concerning the spatial organisation and dynamics of transcription in vivo: What is the transcription rate in a native system? What is the distribution of rates? Is the rate constant over the complete gene? How is transcription spatially organised? How can transcription be regulated? The purpose of the study is to advance the understanding of transcription mechanisms in vivo at the level of single molecules.