Abstract
Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. Here, we devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least ∼20% efficiency and with a high initiation rate of ∼1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust and responsive transcription regulation.