Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites

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 × 10 4 M −1 s −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., competitorfree) 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 STATEMENTTranscription 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.. CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/135947 doi: bioRxiv preprint first posted online May. 9, 2017; 3 \body INTRODUCTION Protein-DNA interactions ultimately control all aspects of cellular function through their actions as "transcription factors" (TFs) by regulating gene transcription, folding DNA into chromosomes, and modifying the structure of chromatin; these regulatory and structural functions are often interwoven (1-9). Understanding protein-DNA interaction kinetics is ...

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“…Despite the coarse-grained nature of the model, it has the salient features of facilitated dissociation: two dissociation pathways and extrinsic regulation by competitor proteins. Thus, the reaction scheme describes the unbinding kinetics of various DNA-binding proteins, such as RPA, Fis, and NHP6A (22,42). The protein-DNA complex may be have single fully bound protein (F), a single partially bound protein (P), two partially bound proteins (saturated, or S), or in the unbound state, no bound protein (U).…”

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“…Recent work with a variety of biomolecules, such as transcription factors (20,22,(24)(25)(26)(27)(28)(29), metalloregulators (21,30), chromatin effectors (31), DNA polymerases (32)(33)(34)(35)(36), antibody-antigen complexes (37), and other biological complexes (23,(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) has demonstrated that facilitated dissociation, in which protein dissociation rates depend on ambient protein or other biomolecule concentration, is a widespread phenomenon both in vitro and in vivo. Facilitated dissociation contrasts with classical kinetic models of bimolecular complexes that undergo a simple, binary on/off transitions, and have concentration-dependent association rates and concentration-independent dissociation rates.…”

“…This accelerates (or "facilitates") the unbinding of the original protein from its binding site by inhibiting full rebinding of the partially bound protein and providing an additional dissociation pathway (22-24, 28, 42, 49-54). Since spontaneous unbinding without competitor biomolecules may be slow for these protein-DNA complexes (e.g., < 10 −3 s −1 for the bacterial protein Fis), facilitated dissociation can lead to as much as a 100-fold enhancement in the dissociation rate at physiological protein concentrations (∼ 10 nM for Fis) (20,22).…”