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. Author manuscript; available in PMC: 2017 May 8.
Published in final edited form as: J Mol Biol. 2016 Mar 22;428(9 Pt B):1833–1845. doi: 10.1016/j.jmb.2016.03.016

Figure 5.

Figure 5

Mechanical interplay of gyrase, transcription, and DNA supercoiling investigated using single-molecule methods. (a) Helical tracking of the advancing transcription complex leads to twin supercoiling domains in a constrained DNA duplex [54, 63]. (b) An optical torque wrench assay [64] showed that RNA polymerase stalls due to positive supercoils that accumulate ahead of the enzyme, with a measured stall torque of ~10 pN nm. (c) Single-molecule assay for transcription on tethered constrained circular templates [54]. Fluorescence accumulates during transcription due to an RNA-binding dye. Dynamics can be investigated in the presence of gyrase and/or topoisomerase I. (d) Model for transcriptional bursting based on single-molecule measurements [54]. Topoisomerase I constitutively relieves (−) supercoils behind the transcription complex, leading to the accumulation of (+) supercoils in a constrained chromosomal loop. When gyrase is bound, (+) supercoils are relaxed and transcription can proceed. When gyrase dissociates, accumulated (+) supercoils inhibit transcription, intermittently shutting off gene expression.