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Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation
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.
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.
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2014 (English)In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, 405-+ p.Article in journal, Letter (Refereed) Published
Abstract [en]

Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.

Place, publisher, year, edition, pages
2014. Vol. 46, no 4, 405-+ p.
National Category
Cell Biology Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:uu:diva-225087DOI: 10.1038/ng.2905ISI: 000334510100020OAI: oai:DiVA.org:uu-225087DiVA: diva2:724936
Note

Hammar and Walldén contributed equally to this work.

Available from: 2014-06-13 Created: 2014-05-27 Last updated: 2017-12-05Bibliographically approved
In thesis
1. How precise is cyclic life?: Insights during a single molecule revolution of the bacterial cell cycle.
Open this publication in new window or tab >>How precise is cyclic life?: Insights during a single molecule revolution of the bacterial cell cycle.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial cells reproduce by doubling in size and dividing. The molecular control systems which regulate the cell cycle must do so in a manner which maintains a similar cell size over many generations. A cell can under conditions of fast growth conclude cell cycles in shorter time than the time required to replicate its chromosome. Under such conditions several rounds of replication are maintained in parallel and a cell will inherit replication processes which were initiated by an ancestor. To accomplish this the cell has to initiate and terminate one round of replication during each cell cycle.

To investigate the effects of the cell cycle on gene-regulation in the gut bacterium Escherichia coli, an experimental method combining microfluidics, single molecule fluorescence microscopy and automated analysis capable of acquiring an arbitrary number of complete cell cycles per experiment was developed. The method allowed for the rapid exchange of the chemical environment surrounding the cells. Using this method it was possible to measure the dissociation time of the transcription factor molecule, LacI-Venus, from the native lactose operator sequence, lacO1, and an artificially strong operator, lacOsym, in vivo. The results indicated that regulation of gene-expression from the lactose operon does not occur at equilibrium in living cells. Furthermore, by studying the intracellular location of non-specifically interacting transcription factor molecules it was possible to determine that these do not form long-lived gradients inside the cell as was previously proposed.

By studying the replication machinery and the origin of replication it was found that replication is initiated according to a cell volume per origin which did not vary over different growth conditions. Further, division timing was found to be determined by the initiation event to occur after a fixed time-delay. A consequence of this mode of regulation is an uncertainty relation between the size at birth and the cell cycle time, in which cells will vary more in in the cycle time during conditions of slow growth as compared to fast growth and vary more in birth length during conditions of fast growth as compared to slow growth.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1204
Keyword
E.coli, cell cycle, replication initiation, transcription factor, gene-regulation, single molecule microscopy, microfluidics
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-235546 (URN)978-91-554-9104-8 (ISBN)
Public defence
2014-12-05, BMC A1:111a, Husarg. 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2014-11-13 Created: 2014-11-05 Last updated: 2015-02-03

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Hammar, PetterWalldén, MatsFange, DavidPersson, FredrikBaltekin, ÖzdenUllman, GustafLeroy, PruneElf, Johan

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