title: Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation.
abstract: 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.
authors.affiliation: 1] Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. [2].
authors.forename: Petter
authors.initials: P
authors.lastname: Hammar
authors.affiliation: 1] Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. [2].
authors.forename: Mats
authors.initials: M
authors.lastname: Walldén
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Fange
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Persson
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Ozden
authors.initials: O
authors.lastname: Baltekin
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Gustaf
authors.initials: G
authors.lastname: Ullman
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Prune
authors.initials: P
authors.lastname: Leroy
authors.affiliation: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Elf
mesh_terms: D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009875:Operator Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D056968:Lac Repressors; D014157:Transcription Factors