Teasing apart the contribution of a single variable to the output of a promoter can be tricky because so many factors affect gene expression. Rajkumar et al. achieve this by systematically altering two binding-site sequences of the transcription factor Pho4 in the yeast PHO5 promoter. These altered sites represent a range of binding affinities that were previously determined in vitro. Rajkumar et al. include the binding sites in a library of 209 synthetic promoters, each fused to the mCherry reporter. Each promoter-reporter fusion is integrated into the yeast genome at the same site, which recapitulates the spacing and nucleosome occupancy of the endogenous promoter, and output is quantified at the single-cell level using a microfluidic setup. The authors show that transcription factor binding affinity can be adjusted to finely tune promoter output.
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Binding-site affinities tune promoter output. Nat Methods 10, 933 (2013). https://doi.org/10.1038/nmeth.2667
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DOI: https://doi.org/10.1038/nmeth.2667