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Leroy, Prune
Publications (5 of 5) Show all publications
Jones, D., Unoson, C., Leroy, P., Curic, V. & Elf, J. (2017). Kinetics of dCas9 Target Search in Escherichia Coli. Paper presented at 58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA. Biophysical Journal, 112(3), 314A-314A
Open this publication in new window or tab >>Kinetics of dCas9 Target Search in Escherichia Coli
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2017 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 314A-314AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
CELL PRESS, 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-332758 (URN)000402375600553 ()
Conference
58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06Bibliographically approved
Jones, D., Leroy, P., Unoson, C., Fange, D., Curic, V., Lawson, M. J. & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science, 357(6358), 1420-1423
Open this publication in new window or tab >>Kinetics of dCas9 target search in Escherichia coli
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6358, p. 1420-1423Article in journal (Refereed) Published
Abstract [en]

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-337092 (URN)10.1126/science.aah7084 (DOI)000411880800052 ()28963258 (PubMedID)
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-01-25Bibliographically approved
Hammar, P., Walldén, M., Fange, D., Persson, F., Baltekin, Ö., Ullman, G., . . . Elf, J. (2014). Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation [Letter to the editor]. Nature Genetics, 46(4), 405-+
Open this publication in new window or tab >>Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation
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2014 (English)In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, p. 405-+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.

National Category
Cell Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-225087 (URN)10.1038/ng.2905 (DOI)000334510100020 ()
Note

Hammar and Walldén contributed equally to this work.

Available from: 2014-06-13 Created: 2014-05-27 Last updated: 2017-12-05Bibliographically approved
Hammar, P., Leroy, P., Mahmutovic, A., Marklund, E. G., Berg, O. G. & Elf, J. (2012). The lac repressor displays facilitated diffusion in living cells. Science, 336(6088), 1595-1598
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, p. 1595-1598Article 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
Hammar, P., Walldén, M., Fange, D., Baltekin, Ö., Ullman, G., Persson, F., . . . Elf, J.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)
Keywords
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
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