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lac of Time: Transcription Factor Kinetics in Living Cells
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
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 [en]
gene regulation, transcription factor, lac operon, facilitated diffusion, single molecule imaging
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-198814ISBN: 978-91-554-8674-7 (print)OAI: oai:DiVA.org:uu-198814DiVA: diva2:618056
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
List of papers
1. The lac repressor displays facilitated diffusion in living cells
Open this publication in new window or tab >>The lac repressor displays facilitated diffusion in living cells
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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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-176861 (URN)10.1126/science.1221648 (DOI)000305507500062 ()22723426 (PubMedID)
External cooperation:
Available from: 2012-06-26 Created: 2012-06-26 Last updated: 2017-12-07Bibliographically approved
2. Transcription factor dissociation measurements using single molecule chase in living cells
Open this publication in new window or tab >>Transcription factor dissociation measurements using single molecule chase in living cells
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(English)Manuscript (preprint) (Other academic)
Keyword
Escherichia coli, Gene regulation, Transcription factor, single molecule imaging
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-198801 (URN)
Available from: 2013-04-25 Created: 2013-04-25 Last updated: 2014-11-05Bibliographically approved
3. LacI follows a helical path while sliding along DNA
Open this publication in new window or tab >>LacI follows a helical path while sliding along DNA
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(English)Manuscript (preprint) (Other academic)
Keyword
Molecular dynamics, Free energy, Umbrella sampling, Facilitated diffusion, Gene regulation, Escherichia coli, Lac Operon, Lac Repressors, Nucleic acids, DNA, Transcription factors, Single molecule imaging
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-198792 (URN)
Available from: 2013-04-25 Created: 2013-04-25 Last updated: 2016-04-21

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