Transcription factor exchange enables prolonged transcriptional bursts.

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.

Time will tell: comparing timescales to gain insight into transcriptional bursting.

Recent imaging studies have captured the dynamics of regulatory events of transcription inside living cells. These events include transcription factor (TF) DNA binding, chromatin remodeling and modification, enhancer-promoter (E-P) proximity, cluster formation, and preinitiation complex (PIC) assembly. Together, these molecular events culminate in stochastic bursts of RNA synthesis, but their kinetic relationship remains largely unclear. In this review, we compare the timescales of upstream regulatory steps (input) with the kinetics of transcriptional bursting (output) to generate mechanistic models of transcription dynamics in single cells. We highlight open questions and potential technical advances to guide future endeavors toward a quantitative and kinetic understanding of transcription regulation.

Transcription factor clusters enable target search but do not contribute to target gene activation.

Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and how it influences gene expression is not well understood. Here, we use quantitative microscopy in living cells to study the regulation and function of clustering of the budding yeast TF Gal4 in its endogenous context. Our results show that Gal4 forms clusters that overlap with the GAL loci. Cluster number, density and size are regulated in different growth conditions by the Gal4-inhibitor Gal80 and Gal4 concentration. Gal4 truncation mutants reveal that Gal4 clustering is facilitated by, but does not completely depend on DNA binding and intrinsically disordered regions. Moreover, we discover that clustering acts as a double-edged sword: self-interactions aid TF recruitment to target genes, but recruited Gal4 molecules that are not DNA-bound do not contribute to, and may even inhibit, transcription activation. We propose that cells need to balance the different effects of TF clustering on target search and transcription activation to facilitate proper gene expression.

Following the tracks: How transcription factor binding dynamics control transcription.

Transcription, the process of copying genetic information from DNA to messenger RNA, is regulated by sequence-specific DNA-binding proteins known as transcription factors (TFs). Recent advances in single-molecule tracking (SMT) technologies have enabled visualization of individual TF molecules as they diffuse and interact with the DNA in the context of living cells. These SMT studies have uncovered multiple populations of DNA-binding events characterized by their distinctive DNA residence times. In this perspective, we review recent insights into how these residence times relate to specific and non-specific DNA binding, as well as the contribution of TF domains on the DNA-binding dynamics. We discuss different models that aim to link transient DNA binding by TFs to bursts of transcription and present an outlook for how future advances in microscopy development may broaden our understanding of the dynamics of the molecular steps that underlie transcription activation.