Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation
Fig 2
DNA binding time of p65 DNA affinity mutants.
(A) Single particle tracking (SPT) was performed after TNF-α stimulation and p65-Halo translocation into the nucleus. All recorded trajectories were filtered based on a spatial threshold established using an immobile control (Histone subunit H2B, see S3 Fig). After filtering out mobile molecules (i) and correction of photobleaching, the DNA binding time could be estimated from the length of each individual trajectory. (B) Schematic overview of p65 affinity mutants. (C) Normalized survival probability plots (1-CDF plot) of the DNA-bound fraction for p65-WT as well as the DNA affinity mutants KKAA and KKRR. The distributions were fitted using a bi-exponential function revealing the fast (tbfast) and slow (tbslow) DNA binding times. (D) Summary of the obtained fitting parameters together with the relative DNA dissociation constant KD for each construct. The pie chart shows the fraction of events associated to the fast (grey) or slow binding time. Kon* was obtained from single step displacement histograms as described in Methods. While all constructs exhibit similar kon* as well as tbfast, the slow binding time tbslow correlates with the DNA affinity.