The overall long-range objective is to quantitatively understand the mechanics of transcriptional control through DNA looping. This will not only elucidate the relationship between the structure, dynamics and function of such regulatory loops, but also improve our understanding of a wide range of fundamental life processes in which proteins interact with multiple sites on a DNA molecule, such as site-specific recombination and the regulation of replication.For this aim, a simplified model system for DNA looping based on the lac repressor and the lac operon in E. coli will be studied in vitro in a series of single-molecule experiments. Specifically, state-of-the-art single-molecule techniques, such as an optical-tweezer based femtoNewton force spectroscopy technique and total-internal-reflection fluorescence microscopy methods will be adapted and further improved to allow the measurement of forces that are associated with the loop formation process with femtoNewton sensitivity and millisecond time resolution while the substrate DNA is subjected to mechanical constraints that are ubiquitous in a living cell, such as tension, twist and supercoiling. The dependence of protein-mediated loop formation rates on these mechanical features will be studied, and quantitatively interpreted in the framework of statistical mechanics of DNA molecules. In the end, it will be attempted to directly control transcription by mechanically opening and closing the repressor loop through the application of tension. This will test current models of how DNA looping regulates transcription and explore the role of mechanical constraints on this important process. This will help to bridge the gap between oversimplified models for DNA looping such as in-vitro DNA ring cyclization, and transcriptional repression in a living cell, which is currently inaccessible to a quantitatively accurate theoretical description. On the side, the instrument development efforts will pave the way for a multitude of other ultra-sensitive single-molecules studies in fields as diverse as the dynamics of protein- and RNA folding or improved studies of molecular motors.
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