Print Email Facebook Twitter Single-molecule visualization of twin-supercoiled domains generated during transcription Title Single-molecule visualization of twin-supercoiled domains generated during transcription Author Janissen, R. (TU Delft BN/Bionanoscience) Barth, R. (TU Delft BN/Cees Dekker Lab) Polinder, M.L. (TU Delft BN/Cees Dekker Lab) van der Torre, J. (TU Delft BN/Cees Dekker Lab) Dekker, C. (TU Delft BN/Cees Dekker Lab) Department BN/Bionanoscience Date 2024 Abstract Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription. To reference this document use: http://resolver.tudelft.nl/uuid:8884bec9-7326-436a-8723-86870fb14c87 DOI https://doi.org/10.1093/nar/gkad1181 ISSN 0305-1048 Source Nucleic acids research, 52 (4), 1677-1687 Part of collection Institutional Repository Document type journal article Rights © 2024 R. Janissen, R. Barth, M.L. Polinder, J. van der Torre, C. Dekker Files PDF gkad1181.pdf 7.3 MB Close viewer /islandora/object/uuid:8884bec9-7326-436a-8723-86870fb14c87/datastream/OBJ/view