Molecular interconnections between transcription, translation and DNA repair in bacteria

This proposal brings together the fields of bacterial transcription, translation and DNA repair with state-of-the art single-molecule biophysics in vitro and in vivo, biochemistry and structural biology to address a central but understudied question in biology: how are different macromolecular processes interconnected and functionally coupled?

I aim to elucidate this by investigating the crosstalk between central processes in bacterial gene expression: transcription, translation and DNA repair.First, to understand what determines whether transcription is coupled to translation, we will reconstitute the complete transcription-translation system in vitro and image the process in real-time using cutting-edge multi-color single-molecule fluorescence experiments.

We will support our dynamic single-molecule data with structural information from cryo-electron tomography.

In order to verify that our in vitro data also have physiological relevance, we will electroporate reconstituted and labelled RNAP/ribosome complexes into live E. coli cells for multi-color single-molecule imaging also in vivo. Second, we will reconstitute the transcription-coupled DNA nucleoside excision repair pathway in vitro.

Integrating multi-color single-molecule fluorescence with optical tweezer assays, will allow us to resolve a controversial point on how transcription-coupled DNA repair starts, comparing the established concept of Mfd-initiation with the newly proposed UvrD/RNAP-mediated lesion recognition.Finally, we will develop single-molecule experiments for integrating all three processes, specifically transcription, translation and DNA repair, into a single system to quantify cooperativity and competition between individual components and processes.Overall, this proposal will provide quantitative and mechanistic understanding of how central processes to gene expression are functionally interconnected, setting a new benchmark for examining complex biological systems in context.