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Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites

Ramsey I. Kamar, Edward J. Banigan, Aykut Erbas, Rebecca D. Giuntoli, Monica Olvera de la Cruz, Reid C. Johnson, John F. Marko
doi: https://doi.org/10.1101/135947
Ramsey I. Kamar
aDepartment of Molecular Biosciences, Northwestern University, Evanston, IL, USA
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Edward J. Banigan
bDepartment of Physics and Astronomy, Northwestern University, Evanston, IL, USA
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Aykut Erbas
cDepartment of Materials Science and Engineering, Northwestern University, Evanston, IL, USA
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Rebecca D. Giuntoli
aDepartment of Molecular Biosciences, Northwestern University, Evanston, IL, USA
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Monica Olvera de la Cruz
bDepartment of Physics and Astronomy, Northwestern University, Evanston, IL, USA
cDepartment of Materials Science and Engineering, Northwestern University, Evanston, IL, USA
dDepartment of Chemistry, Northwestern University, Evanston, IL, USA
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Reid C. Johnson
eDepartment of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
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John F. Marko
aDepartment of Molecular Biosciences, Northwestern University, Evanston, IL, USA
bDepartment of Physics and Astronomy, Northwestern University, Evanston, IL, USA
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ABSTRACT

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

SIGNIFICANCE STATEMENT Transcription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.

Author Contributions Ramsey I. Kamar

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Edward J. Banigan

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Aykut Erbas

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Rebecca D. Giuntoli

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Monica Olvera de la Cruz

designed research, performed research, wrote the paper

Reid C. Johnson

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

John F. Marko

designed research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted May 09, 2017.
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Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites
Ramsey I. Kamar, Edward J. Banigan, Aykut Erbas, Rebecca D. Giuntoli, Monica Olvera de la Cruz, Reid C. Johnson, John F. Marko
bioRxiv 135947; doi: https://doi.org/10.1101/135947
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Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites
Ramsey I. Kamar, Edward J. Banigan, Aykut Erbas, Rebecca D. Giuntoli, Monica Olvera de la Cruz, Reid C. Johnson, John F. Marko
bioRxiv 135947; doi: https://doi.org/10.1101/135947

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