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

Summary The canonical B-form DNA helix and its protein interactions are well-characterized, yet the behavior of torsionally constrained DNA, ubiquitous in cells, remains underexplored. Using cryo-electron tomography (cryo-ET), we 3D-reconstructed entire negatively supercoiled DNA substrates with bound RNA polymerase (RNAP), revealing DNA supercoiling conformational diversity and its interplay with molecular motors. RNAP and DNA-binding proteins like dCas9 preferentially localize at plectoneme apices during transcription, acting as torsional blocks. Together, dCas9 and RNAP on opposing plasmid apices can segregate "twin-supercoiling domains" without the need for external DNA end-tethering. The generation of twin domains reveals as regions of reduced supercoiling and the presence of multiple transcribing RNAP complexes. Negative supercoiling and apex localization of RNAP favor initiation but disfavor elongation, leading to slow-moving RNAP clusters. Topoisomerase I relieves RNAP pauses by removing them from apical constraints; the resulting RNAP load-and-release process from the apex provides a molecular mechanism for the "transcriptional bursting" phenomenon.