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What matters for lac repressor search in vivo-sliding, hopping, intersegment transfer, crowding on DNA or recognition?
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.
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.
2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, 3454-3464 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated which aspects of transcription factor DNA interactions are most important to account for the recent in vivo search time measurements for the dimeric lac repressor. We find the best agreement for a sliding model where non-specific binding to DNA is improbable at first contact and the sliding LacI protein binds at high probability when reaching the specific O-sym operator. We also find that the contribution of hopping to the overall search speed is negligible although physically unavoidable. The parameters that give the best fit reveal sliding distances, including hopping, close to what has been proposed in the past, i.e. similar to 40 bp, but with an unexpectedly high 1D diffusion constant on non-specific DNA sequences. Including a mechanism of inter-segment transfer between distant DNA segments does not bring down the 1D diffusion to the expected fraction of the in vitro value. This suggests a mechanism where transcription factors can slide less hindered in vivo than what is given by a simple viscosity scaling argument or that a modification of the model is needed. For example, the estimated diffusion rate constant would be consistent with the expectation if parts of the chromosome, away from the operator site, were inaccessible for searching.

Place, publisher, year, edition, pages
2015. Vol. 43, no 7, 3454-3464 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-256544DOI: 10.1093/nar/gkv207ISI: 000354722500012PubMedID: 25779051OAI: oai:DiVA.org:uu-256544DiVA: diva2:826372
Available from: 2015-06-25 Created: 2015-06-24 Last updated: 2017-12-04Bibliographically 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)
Opponent
Supervisors
Available from: 2015-10-16 Created: 2015-10-02 Last updated: 2016-09-09

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Mahmutovic, AnelBerg, Otto G.Elf, Johan

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