Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells

Vukojevic, V. and Papadopoulos, D. K. and Terenius, L. and Gehring, W. J. and Rigler, R.. (2010) Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells. Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, H. 9. pp. 4093-4098.

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Official URL: http://edoc.unibas.ch/dok/A5842530

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Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to ``titrate`` the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K(d)) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Cell Biology (Gehring)
UniBasel Contributors:Gehring, Walter Jakob
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:National Academy of Sciences
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:08 Jun 2012 06:56
Deposited On:08 Jun 2012 06:49

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