Chromosomes carry genetic instructions that allow cells to grow, respond to the environment, and pass information to future generations. Just as the function of a protein is understood through its amino acid sequence, three-dimensional structure, and conformational dynamics, a complete understanding of chromosome function requires knowledge of DNA sequence, chromosome structure, and dynamic behavior. Modern sequencing and imaging methods have transformed the ability to read genomes and capture snapshots of chromosome architecture, but much less is known about how different regions of the chromosome move inside living cells. The project will address this gap in knowledge by creating a genome-scale view of chromosome dynamics in the bacterium Escherichia coli and establishing physical principles that explain why different chromosomal regions move in different ways. The outcomes have broad implications for the U.S. national interest by promoting fundamental discovery at the interface of physics and biology, strengthening quantitative approaches in biotechnology, and helping build a foundation for future efforts to predict and engineer genome function. Integrated education and outreach activities will train students across multiple levels in quantitative biophysics, broaden access to research experiences, disseminate protocols and analysis tools, and highlight the contributions of physicists to biology and medicine, including Nobel laureate and Illinois alumna Rosalyn Yalow. The project will combine high-resolution single-molecule tracking, MINFLUX super-resolution microscopy, quantitative polymer-physics modeling, and in vitro reconstitution to decode how active biological processes and passive physical constraints shape bacterial chromosome dynamics. In living cells, the project will measure fast-timescale motion of many genomic loci to build a high-resolution atlas of chromosome motion. These measurements will be combined with complementary genomic and cellular da