Probing temporal sequences and molecular mechanisms of transcription initiation by single molecule fluorescence microscopy in living eukaryotic cells

• Applicant: Professor Dr. Christof Gebhardt
• Subject Area: Biochemistry ; Biophysics
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258796734

The initial binding of a transcription factor to its target site on DNA triggers a series of events that finally lead to the assembly of the transcription machinery and the production of RNA. While for many genes the molecular species participating in the initiation pathway are known, detailed kinetic information on their interactions with one another and with DNA is largely missing. Thus, key questions concerning the temporal coordination of transcription initiation, the flow of information from transcription factor binding to the onset of transcription and the efficiency of this process remain unanswered. This proposal aims at understanding important dynamical aspects of gene transcription and its initiation by both measuring and directly manipulating cellular dynamics. We will use single molecule fluorescence microscopy techniques such as reflected light sheet microscopy to monitor the actions of individual biomolecules involved in transcription initiation in the nuclei of living mammalian cells and extract kinetic information on their interplay with interaction partners. In the first part of the project we plan to develop artificial transcription factors with a controllable residence time on DNA and measure how the temporal profile of gene transcription depends on this parameter. In the second part, we will study the dynamics of essential basal transcription factors and pathways of their assembly into the transcription machinery.

• DFG Programme: Research Grants
• Participating Persons: Professor Dr. Holger Barth; Professor Dr. Jens Michaelis