Promoter variants and experimental setup. (A) Illustration of hypothesized promoter dynamics induced by two distinct DNA-encoded strategies for increasing expression and a matched stochastic simulation of promoter switching, transcription, and translation (right panel) for each strategy. For a simplified model in which promoters transition at some rate between transcriptionally inactive and active states (top), we hypothesize that addition of nucleosome disfavoring elements such as poly(dA:dT) tracts would increase the accessibility of the transcription factor binding site, thereby reducing the time that a factor molecule spends in search of its site (middle). In contrast, we hypothesize that increasing the affinity of a factor binding site would reduce the factor's dissociation rate. Note that both types of sequence changes result in a higher mean expression over the cell population, but with distinct hypothesized effects on promoter dynamics. Using the Gillespie algorithm, we simulate the kinetic scheme in three scenarios. In the simulation runs, we record, as a function of time, the promoter state (black–red line), mRNA levels (blue line), protein levels (black line), and the protein production rate (green line), which is the derivative of the protein levels with respect to time. In addition, we record protein production bursts (red line), when production is positive. The “normal” promoter (top) represents the reference point for the parameter changes. The fast promoter (middle) has an increased kon to simulate an increase in promoter accessibility [added poly(dA:dT)]. The “slow” promoter (bottom) has a decreased koff (with respect to the normal promoter) to simulate an increase in TF binding site affinity. The three example runs shown illustrate that although both parameters can increase the overall expression level, kon increases the frequency of production bursts, while koff increases the length of the bursts. (B) Illustration of promoter variants used in this study. All promoters are genomically integrated upstream of a yellow fluorescent protein (YFP) reporter and into a region that also contains an mCherry fluorescent protein driven by a constant TEF2 promoter. Promoter variants differ in the presence and length of two poly(dA:dT) tracts and in the affinity of the transcription factor binding site for either the Gcn4 or Gal4 transcriptional activators. (C) Representative YFP time-lapse microscopy images of four promoter variants, imaged in a microfluidic platform that supplies a continuous flow of medium. Each cell was followed over time, and its lineage, YFP, and mCherry signal intensity were extracted (Methods). (D) Single-cell flow cytometry data collected for the corresponding promoter variants from C, shown as a histogram of normalized YFP values (top) and as a scatterplot of YFP (x-axis) against mCherry fluorescence (bottom).