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Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-9804-5009
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-6084-0197
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 49, 19796-19801 p.Article in journal (Refereed) Published
Abstract [en]

Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k(B)T for sliding and 12 kBT for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.

Place, publisher, year, edition, pages
2013. Vol. 110, no 49, 19796-19801 p.
Keyword [en]
facilitated diffusion, lac operon, lac repressors, gene regulation
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-213898DOI: 10.1073/pnas.1307905110ISI: 000327744900041OAI: oai:DiVA.org:uu-213898DiVA: diva2:683744
Available from: 2014-01-06 Created: 2014-01-05 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Reaction-Diffusion kinetics of Protein DNA Interactions
Open this publication in new window or tab >>Reaction-Diffusion kinetics of Protein DNA Interactions
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transcription factors need to rapidly find one specific binding site among millions of nonspecific sites on the chromosomal DNA. In this thesis I use various aspects of reaction-diffusion theory to investigate the interaction between proteins and DNA and to explain the searching, finding and binding to specific operator sites. Using molecular dynamics methods we calculate the free energy profile for the model protein LacI as it leaves a nonspecific stretch of DNA and as it slides along DNA. Based on the free energy profiles we estimate the microscopic dissociation rate constant, kdmicro ~1.45×104s-1, and the 1D diffusion coefficient, D1 ~ 0.05-0.29 μm2s-1 (2-40μs to slide 1 basepair (bp)). At a non-atomistic level of detail we estimate the number of microscopic rebindings before a macroscopic dissociation occurs which leads to the  macroscopic residence time, τDmacro ~ 48±12ms resulting in a in vitro sliding length estimate of 135-345bp.

When we fit the DNA interaction parameters for in vivo conditions to recent single molecule in vivo experiments we conclude that neither hopping nor intersegment transfer contribute to the target search for the LacI dimer, that it appears to bind the specific Osym operator site as soon as it slides into it, and that the sliding length is around 40bp in the cell. The estimated in vivo D1 ~ 0.025 μm2s-1 is higher than expected from estimates of D1 based on viscosity and the atomistic simulations. Surprisingly, we were also forced to conclude that the nonspecific association for the LacI dimer appeared reaction limited which is in conflict with the free energy profile. This inconsistency is resolved by allowing for steric effects. Using reaction-diffusion theory and simulations we show that an apparent reaction limited association can be diffusion limited if geometry and steric effects are taken into account. Furthermore, the simulations show that a protein binds ~2 times faster to a DNA molecule with a helical reactive patch than to a stripe patch running along the length of the DNA. This facilitated binding has a direct impact on the search time especially in the presence of other DNA binding proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1299
Keyword
umbrella sampling, molecular dynamics, RDME, PDE, sliding, intersegment transfer, hopping, sterics, intersegment transfer
National Category
Biophysics Bioinformatics and Systems Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-263527 (URN)978-91-554-9360-8 (ISBN)
External cooperation:
Public defence
2015-11-06, C8:301, Husargatan 3, Uppsala, 13:00 (English)
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Supervisors
Available from: 2015-10-16 Created: 2015-10-02 Last updated: 2016-09-09

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Mahmutovic, AnelBerg, Otto G.Hammar, Pettervan der Spoel, DavidFange, DavidElf, Johan

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Marklund, Erik G.Mahmutovic, AnelBerg, Otto G.Hammar, Pettervan der Spoel, DavidFange, DavidElf, Johan
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