Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

Protein interactions with canonical B-form DNA are well-characterized, yet the effect on these interactions of torsionally constrained DNA—ubiquitous in cells—remains underexplored. Using cryo-electron tomography (cryo-ET), we 3D-reconstructed entire negatively supercoiled DNA substrates bound to active RNA polymerase (RNAP), revealing diverse DNA supercoiling conformations and their interplay with transcription. RNAP preferentially localizes at plectoneme apices in a swiveled, pause-prone state. RNAP, along with other DNA-melting proteins like dCas9, can act as torsional roadblocks that segregate “twin-supercoiling domains” during active transcription, independent of external DNA/RNAP tethering. Co-transcribing RNAPs further intensify this domain separation: tandem oriented RNAPs relieve negative supercoiling more effectively than opposing ones, promote greater RNAP accumulation and enhanced elongation, both in vitro and in vivo. Topoisomerase I relieves torsional stress and facilitates RNAP escape from apical stalls, thereby supporting apical transcription regulation. Together, these findings support a load-and-release mechanism at plectoneme apices that may underlie supercoiling-dependent transcriptional bursting.