Rajkumar, A.S. et al. Nat. Genet. 10.1038/ng.2729 (18 August 2013).

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.