Mechanisms of complex transcriptional processes and assemblies in bacteria

Transcription is vital for the lifestyle of bacteria. To achieve adaptation in a host, or survive under hostile conditions, bacteria employ a complex network of responses centred around transcriptional control of related genes, some of which give rise to virulence and antibiotic resistance. However, despite progress in structural and single-molecule biophysical studies of transcription, we are far from understanding how transcription operates inside a bacterial cell, which features an enormously complex and fluctuating environment impossible to reconstitute in vitro. We will address this complexity using cutting-edge single-molecule methodologies we have been pioneering over the past decade. We will perform direct, real-time monitoring of single molecules of RNA polymerase and transcription factors as they go through the transcription cycle inside living cells, as well as high- resolution multiplexed imaging of large transcriptional clusters that may represent biomolecular condensates forming via liquid-liquid phase separation. Using our advanced toolbox and complementary approaches, we will elucidate controversial kinetic aspects of the transcription cycle, resolve the structure, composition, and physical nature of the large transcriptional clusters, and identify functions of these clusters in promoter search and transcription re-initiation. Our work will revolutionise the understanding of bacterial transcription, while enabling applications in biotechnology, therapeutics and synthetic biology.