Live‐cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting

Abstract

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single‐molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation. A combination of single‐molecule in vitro and in vivo imaging to study the interplay between transcription factor dwell time, nucleosome binding and transcriptional bursting at the galactose‐responsive genes in budding yeast suggests a model for RNA polymerase initiation during transcriptional bursts. Single‐molecule approaches to correlate Gal4 transcription factor binding with target gene transcriptional bursting kinetics suggests that multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA.