Abstract
Transcription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA–protein interactions not only by preventing DNA–protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.
Highlights
Initiation of eukaryotic gene expression involves transcription factor (TF) binding to DNA-target sites at gene promoters within chromatin [1,2]
To confirm that the Cy3 signal acquired was from immobilized DNA molecules instead of any possible background fluorescence in the flow cell, a Cy5 fluorophore was attached on the same DNA further away from the LexA-target site (Figure 1D) so that only the signal from a Cy3 that colocalized with a Cy5 was selected for analysis
These values agreed with the EMSA and ensemble Protein Induced Fluorescence Enhancement (PIFE) measurements of LexA binding to DNA (Figure 2C)
Summary
Initiation of eukaryotic gene expression involves transcription factor (TF) binding to DNA-target sites at gene promoters within chromatin [1,2]. TFs target specific genes by binding particular DNA sequences with high affinities that are quantified by the dissociation constant, KD = koff/kon (Figure 1A). The KD is the characteristic concentration for binding and can be determined experimentally by measuring S0.5, the concentration of TF at which 50% of the target DNA sequence is bound. The KD is typically between nanomolar and picomolar for DNA-binding TFs. The KD is typically between nanomolar and picomolar for DNA-binding TFs This is usually achieved by having relatively long resident times on, and slow dissociation rates from the target sequence. TFs can have residence times of about an hour at their DNArecognition sequence [13,14], which implies dissociation rates as low as 10À4sÀ1. The mechanisms by which TF dissociation is dramatically accelerated remain unknown [20,21]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
