Synthetic Biology – measuring non-linearities in gene expression

Gene expression is complicated. Even when the promoters are identical, each gene has a unique TF dose-response curve. We found that genes intrinsically buffer protein levels from changes in promoter activity.

 

Promoter Activity Buffering Reduces the Fitness Cost of Misregulation

Miquel Àngel Schikora-Tamarit, Guillem Lopez-Grado i Salinas, Carolina Gonzalez-Navasa, Irene Calderón ,Xavi Marcos-Fa, Miquel Sas & Lucas B. Carey

Cell Reports .   DOI:https://doi.org/10.1016/j.celrep.2018.06.059

  • Systematic replacement of native promoters by a constant synthetic inducible promoter
  • Even when the promoters are identical, each gene has a unique TF dose-response curve
  • Gene-specific DRCs are not from differences in translation, or mRNA or protein stability
  • Most genes intrinsically buffer protein levels from changes in promoter activity.

 

Organisms regulate gene expression through changes in the activity of transcription factors (TFs). In yeast, the response of genes to changes in TF activity is generally assumed to be encoded in the promoter. To directly test this assumption, we chose 42 genes and, for each, replaced the promoter with a synthetic inducible promoter and measured how protein expression changes as a function of TF activity. Most genes exhibited gene-specific TF dose-response curves not due to differences in mRNA stability, translation, or protein stability. Instead, most genes have an intrinsic ability to buffer the effects of promoter activity. This can be encoded in the open reading frame and the 3′ end of genes and can be implemented by both autoregulatory feedback and by titration of limiting trans regulators. We show experimentally and computationally that, when misexpression of a gene is deleterious, this buffering insulates cells from fitness defects due to misregulation.

(A) A schematic view of the question. Promoter elements determine the response to transcription factor (TF) activity in gene expression. However, it is unknown which is the contribution of the open reading frame (ORF) and 3′ end regions.
(B) As an example, β-estradiol activates the engineered zinc-finger TF Z3 (McIsaac et al., 2013). We engineered a yeast strain library of 42 genes, replacing the native 5′ regulatory region with a synthetic β-estradiol promoter driving an mCherry-protein tag, while GFP remains as a pathway-specific reporter of promoter activity of each gene.
(C) Three example genes and a control strain (that lacks an mCherry-protein tag) show similar TF activity-versus-GFP expression profiles (top). However, TF-versus-mCherry-protein expression profiles (bottom) are diverse.
(D) Each gene exhibits a unique GFP (as a proxy for promoter activity)-versus-mCherry (as a proxy for protein level) profile, despite having identical promoters. Three example genes illustrate the diverse shapes of the curves.