Single-Molecule Imaging Reveals Transcription-Driven Supercoiling in Unconstrained DNA

In vitro studies of supercoiling dynamics have relied on externally applied force to twist constrained DNA. It is thus unknown whether transcription alone can generate supercoiling in topologically unconstrained DNA, and whether RNAP complexes could act as the topological barriers required to confine this stress. Here, using single-molecule imaging of 20k base pair long double strand DNA, we reveal that RNAP dynamically generates and confines supercoiling in unconstrained DNA. We demonstrate that multiple transcription events create transient topological domains, which allow plectonemes to stabilize between them; in contrast, a single transcription event does not lead to plectoneme formation. Furthermore, we show that this transcription-induced supercoiling is modulated by topoisomerase activity. Crucially, by observing that RNAP itself provides sufficient topological barriers, we establish that transcription-induced supercoiling is an inherently localized phenomenon. These findings redefine the physical basis of transcription-coupled supercoiling, providing a new mechanistic framework for modeling gene regulation based on local topology, with broader implications for genome editing and genome evolution.