Abstract
Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.
Highlights
Cells respond to their environment through gene regulation involving protein transcription factors
These results demonstrate a functional link between the localization of Mig1 and Msn2 clusters, and the transcribed mRNA from their target genes
We observe that the repressor protein Mig1 forms clusters which, upon extracellular glucose detection, localize dynamically from the cytoplasm to bind to locations consistent with promoter sequences of its target genes
Summary
Cells respond to their environment through gene regulation involving protein transcription factors. These proteins bind to DNA targets of a few tens of base pairs (bp) length inside ~500-1,000bp promoter sequences to repress/activate expression, involving single [1] and multiple [2] factors, resulting in the regulation of target genes. Target finding may involve heterogeneous mobility including nucleoplasmic diffusion, sliding and hops along DNA up to ~150bp, and even longer jumps separated by hundreds of bp called intersegment transfer [4,5,6]. BY4741 MSN2-mKO2::LEU2 MIG1(Δaa36-91)mCherry::spHIS5 GSY1-24xPP7::KANMX msn4Δ mig2Δnrg1::HPHMX nrg2::Met. Hohmann collection [64] This study [16] This study This study This study [18] [18]
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