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Transcription factor dissociation measurements using single molecule chase in living cells
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nyckelord [en]
Escherichia coli, Gene regulation, Transcription factor, single molecule imaging
Nationell ämneskategori
Naturvetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-198801OAI: oai:DiVA.org:uu-198801DiVA, id: diva2:617990
Tillgänglig från: 2013-04-25 Skapad: 2013-04-25 Senast uppdaterad: 2014-11-05Bibliografiskt granskad
Ingår i avhandling
1. lac of Time: Transcription Factor Kinetics in Living Cells
Öppna denna publikation i ny flik eller fönster >>lac of Time: Transcription Factor Kinetics in Living Cells
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2013. s. 74
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1046
Nyckelord
gene regulation, transcription factor, lac operon, facilitated diffusion, single molecule imaging
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Molekylär bioteknik
Identifikatorer
urn:nbn:se:uu:diva-198814 (URN)978-91-554-8674-7 (ISBN)
Disputation
2013-06-14, B42, Biomedicinskt centrum, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-05-22 Skapad: 2013-04-25 Senast uppdaterad: 2013-08-30Bibliografiskt granskad

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