Genomic DNA is a crowded track shared by many motor proteins and decorated with a plethora of roadblocks. Proper traffic management is critical to the maintenance of genome integrity and control of gene activity. However, the molecular rules and functional consequences of motor collisions and roadblock encounters remain poorly understood. We have built an experimental pipeline—leveraging biochemical reconstitution and single-molecule visualization—to interrogate these dynamic events in real time. Our findings suggest that collisions among DNA-based motors, generally thought to be deleterious to genome stability, can be harnessed to control the output of gene expression.