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The dynamic response of upstream DNA to transcription-generated torsional stress

Nature Structural & Molecular Biology volume 11, pages 1092–1100 (2004)Cite this article

Abstract

The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.

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Figure 1: High-resolution mapping of supercoil-dependent chemical modification within the FUSE.
Figure 2: Two-dimensional agarose gel analysis of supercoil-dependent DNA structural transitions in +FUSE and −FUSE midi-circles.
Figure 3: FUSE melting as a torsional strain gauge.
Figure 4: An experimental approach to capture and measure dynamic DNA supercoiling during transcription.
Figure 5: Transcription from a single promoter supports supercoiling of upstream DNA.
Figure 6: Influence of the transcription rate on the intensity of supercoiling and FUSE melting.
Figure 7: Binding of FBP to minicircle topoisomers.
Figure 8: FUSE, FBP and real-time regulation of transcription.

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Acknowledgements

We thank L. Liotta, D. Clark, D. Boles and L. Benjamin for critical comments.

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  1. Laboratory of Pathology, National Cancer Institute, Bethesda, 20892-1500, Maryland, USA

    Fedor Kouzine, Juhong Liu, Suzanne Sanford, Hye-Jung Chung & David Levens

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Correspondence to David Levens.

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Supplementary information

Supplementary Fig. 1

Effect of transcription on KMnO4 reactivity at the FUSE element. (PDF 46 kb)

Supplementary Fig. 2

Influence of the viscosity of the reaction media, and nascent transcript length on the intensity of dynamic supercoiling. (PDF 43 kb)

Supplementary Fig. 3

Evanescent nature of the transcriptionally generated stress. (PDF 48 kb)

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Kouzine, F., Liu, J., Sanford, S. et al. The dynamic response of upstream DNA to transcription-generated torsional stress. Nat Struct Mol Biol 11, 1092–1100 (2004). https://doi.org/10.1038/nsmb848

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