Molecular mechanisms and regulation of bacterial transcription in vivo (360G-Wellcome-110164_Z_15_Z)

My overarching question is the elucidation of the molecular mechanisms that living cells employ to regulate the transcription of genes in a precise spatial and temporal manner. Transcription is a fundamental process for all organisms, constituting the first step in gene expression, and controlling the ability of living systems to develop, differentiate, sense their environment and adapt. These herculean tasks require tight regulation mechanisms ensuring that the right genes are expressed at the right time, the right place, and at the levels required. The timing of transcription is controlled during transcription initiation, a complicated process with many transitions involving transient, dynamic complexes formed through protein and DNA conformational changes. This dynamic landscape, which is further complicated in vivo, is challenging to address using current approaches: crystallography provides only static snapshots of stabilized structural states, whereas ensemble biochemistry cannot address the heterogeneous, asynchronous populations of initiation complexes. Systems approaches have aided our in vivo understanding, but since they typically examine cell populations, they cannot report on in vivo kinetics, cell-cell heterogeneity, or transcription dependence on cellular location. My vision is to face these challenges by bringing the power of molecular analysis into the biological context of living cells. This will entail a single-molecule approach that bypasses ense mble averaging and allows direct, real-time visualization of transcription on single genes inside single living cells. Using bacterial transcription as our system, we will answer the following: 1. How does the RNA polymerase melt, transcribe and escape from the promoter during transcription initiation? What are the kinetics of key conformational changes, and how are they regulated by activators and antibiotics? 2. How is the timing of initiation regulated in vivo? How heterogeneous is initi ation in space and time in single cells, and what is the cell-to-cell variability?