DNA surface exploration and operator bypassing during target search
| Autor: | Xuan Zheng, Sebastian Deindl, Laura C. Lehmann, Andrew Emmerich, Otto G. Berg, Kalle Kipper, Elias Amselem, Brad Van Oosten, Magnus Johansson, Emil Marklund, Anton Sabantsev, Johan Elf, Daniel Globisch, Guanzhong Mao |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Operator Regions
Genetic Operator (biology) Rotation Phase (waves) Biophysics Fluorescence correlation spectroscopy Lac repressor 7. Clean energy ENERGY-TRANSFER SINGLE PROTEIN TRANSLOCATION MICROSCOPY DIFFUSION DYNAMICS Substrate Specificity Diffusion 03 medical and health sciences 0302 clinical medicine Fluorescence Resonance Energy Transfer Lac Repressors 030304 developmental biology Physics 0303 health sciences Millisecond Binding Sites Multidisciplinary Rotational diffusion DNA Single Molecule Imaging Biofysik Kinetics Microsecond Spectrometry Fluorescence Förster resonance energy transfer Nucleic Acid Conformation Biological system 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | Nature |
| Popis: | Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA(1-5). 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 mu s. 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. Single-molecule fluorescence resonance energy transfer and real-time confocal laser tracking with fluorescence correlation spectroscopy together characterize how individual lac repressor molecules bypass operator sites while exploring the DNA surface at microsecond timescales. |
| Databáze: | OpenAIRE |
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