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bioRxiv · 10.64898/2026.03.25.714350

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

Zhang, M.·Canari-Chumpitaz, C.·Liu, J.·Onoa, B.·de Cleir, S.·Cheng, E.·Requejo, K. I.·Bustamante, C.
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Abstract

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.

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Zhang, M., Canari-Chumpitaz, C., Liu, J., Onoa, B., de Cleir, S., Cheng, E., Requejo, K. I., Bustamante, C.. 2026-03-26. Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET. https://doi.org/10.64898/2026.03.25.714350

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