DNA surface exploration and operator bypassing during target search

Abstract

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on nonspecific DNA [1][2][3][4][5] . Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 µs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.Sequence-specific binding and recognition of DNA target sites by proteins such as transcription factors, polymerases, and DNA-modifying enzymes is at the core of cellular information processing. However, the 'target search problem' of how to rapidly yet accurately find a specific target sequence remains incompletely understood. One aspect of preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Surface condensation of a pioneer transcription factor on DNA

“…However, we can speculate on the implications of our work for the process by which transcription factors find their target sites. Our observations are consistent with previous suggestions that, at the molecular scale, transcription factors could undergo a 1D diffusive search along DNA, for example in the adsorbed state [55][56][57][58][59] ( Fig. 2c and Extended Data Fig.…”

Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time

“…Recently, LacI was observed to hop along the double helix (Marklund et al, 2020). This feature together with negative supercoiling is probably key for the protein to efficiently locate a binding site, contact a secondary binding site, and maintain a constant ratio of looped versus unlooped states in the various states of tension and torsion that likely develop during the cell cycle.…”

DNA Manipulation and Single-Molecule Imaging

“…Single-molecule FRET (smFRET) facilitates measurement for the dynamic interactions and behavior between a fluorescently labeled donor and an acceptor, such as protein-protein and protein-DNA [135][136][137][138][139] interactions. In addition, by labeling with a donor and an acceptor at different sites on the same single molecule, the conformation changes within a single molecule are allowed to be observed [140,141].…”