DNA surface exploration and operator bypassing during target search

Emil Marklund, Brad Oosten, Guanzhong Mao, Elias Amselem, Kalle Kipper, Anton Sabantsev, Andrew Emmerich, Daniel Globisch, Xuan Zheng, Laura C. Lehmann, Otto G. Berg, Magnus Johansson, Johan Elf () and Sebastian Deindl ()
Additional contact information
Emil Marklund: Uppsala University
Brad Oosten: Uppsala University
Guanzhong Mao: Uppsala University
Elias Amselem: Uppsala University
Kalle Kipper: Uppsala University
Anton Sabantsev: Uppsala University
Andrew Emmerich: Uppsala University
Daniel Globisch: Uppsala University
Xuan Zheng: Uppsala University
Laura C. Lehmann: Uppsala University
Otto G. Berg: Uppsala University
Magnus Johansson: Uppsala University
Johan Elf: Uppsala University
Sebastian Deindl: Uppsala University

Nature, 2020, vol. 583, issue 7818, 858-861

Abstract: Abstract Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1–5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT–FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT–FCS data shows that LacI hops one or two grooves (10–20 bp) every 200–700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein–DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT–FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

Date: 2020
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DOI: 10.1038/s41586-020-2413-7

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