Deciphering molecular and kinetic bases of transcription with single molecule approaches

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Transcription is vital to the cell. Processes such as homeostasis, growth and differentiation require well-balanced protein numbers, wherefore temporally precise regulation of transcription is important. For most genes, mRNA is produced in bursts of multiple transcripts, followed by inactive periods without transcription. Numerous regulatory traits have been observed to influence such transcriptional bursting, including binding of activating transcription factors (TFs), the interplay with co-activators, and enhancer-promoter interactions. However, the mechanistic and kinetic details of how these traits regulate properties of transcriptional bursts such as burst frequency, size and duration, are unclear or missing.With this proposal we aim at understanding several molecular and kinetic aspects of transcription regulation. We plan to manipulate mechanistic and kinetic properties of regulatory traits within the cell, such as the time a TF spends bound to its target site, the number of target sites at the proximal enhancer, or the distance between distal enhancer and promoter, using artificial TFs and reporter genes. We will characterize altered properties and monitor the changes to co-activator interaction and mRNA production by using single molecule fluorescence imaging and single transcript visualization in live and fixed cells. Overall, our approach will yield comprehensive quantitative insight into the regulation of transcription kinetics.