Single-molecule visualization of twin-supercoiled domains generated during transcription

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 within vitroreal-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.

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step

“…However, it is also plausible that supercoiling accumulates within a subset of (sub-)TADs ( 66 ) which could account for the increased intra-TAD contact frequency ( 65 ). While the accumulation of supercoils within TADs has so far been thought to be due to transcription-mediated supercoiling ( 65, 67, 68 ), this could also be the result of cohesin-mediated loop extrusion itself. The accumulation of supercoiling within TADs can contribute to their insulating property ( 69 ) as well as to the facilitation of long-range contacts between genomic loci within a TAD, e.g.…”