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The lac repressor displays facilitated diffusion in living cells
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.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.ORCID iD: 0000-0002-9804-5009
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2012 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 336, no 6088, 1595-1598 p.Article in journal (Refereed) Published
Abstract [en]

Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO(1) operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

Place, publisher, year, edition, pages
2012. Vol. 336, no 6088, 1595-1598 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-176861DOI: 10.1126/science.1221648ISI: 000305507500062PubMedID: 22723426OAI: oai:DiVA.org:uu-176861DiVA: diva2:537271
Available from: 2012-06-26 Created: 2012-06-26 Last updated: 2017-12-07Bibliographically approved
In thesis
1. lac of Time: Transcription Factor Kinetics in Living Cells
Open this publication in new window or tab >>lac of Time: Transcription Factor Kinetics in Living Cells
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules.

   In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding.

   By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path.

   Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1046
Keyword
gene regulation, transcription factor, lac operon, facilitated diffusion, single molecule imaging
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-198814 (URN)978-91-554-8674-7 (ISBN)
Public defence
2013-06-14, B42, Biomedicinskt centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-05-22 Created: 2013-04-25 Last updated: 2013-08-30Bibliographically approved
2. 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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Hammar, PetterLeroy, PruneMahmutovic, AnelMarklund, Erik GBerg, Otto GElf, Johan

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