In this project the PI will study the effects of mechanical constraints on transcription regulation in a model system based on the lactose repressor in E. Coli, using single-molecule techniques. This system is a paradigm for protein-mediated DNA looping a major theme in the regulation of transcription and replication and is biochemically and structurally well characterized. The PI will look into three mechanical constraints that a cellular environment might impose, and their role in protein-mediated DNA looping. First, the PI will study the effect of constraining the angular orientation of the operator sites on the DNA with respect to the protein using DNA constructs with specially designed bends and single-molecule competition assays. These experiments will shed light on the origin of a three-order of magnitude discrepancy in loop lifetimes measure by bulk competition assays and single-molecule tethered-particle microscopy and give insight into how supercoiling enhanced loop formation. Second, the PI will study how noise from a fluctuating cellular environment affects the robustness of the loop formation process, which will help to understand how a loop-forming genetic switch can work reliably despite its extreme sensitivity to small forces. Third, the PI will look at loop formation and breakdown in gels that mimic cellular environments with particular emphasis on understanding why DNA-protein complexes are significantly more stable in gels even when the pore size is so large that the gel should hardly be an obstacle for protein diffusion. The PI will train graduate and undergraduate students in biological physics, using a highly interdisciplinary approach. Special emphasis will be placed on the recruitment of underrepresented minority students and the PI will also engage in a broad range of outreach efforts that include public lectures on topics of general interest in biological physics, and participation in a Single-Molecule Roadshow for high schools.