Evaluating competitive inhibition by gcn5-F221A using a synthetic pPHO5-yECitrine construct.

<p>The <i>gcn5-F221A</i> mutant was expressed with varying promoter strengths in <b>a.</b> the haploid BY4741 <i>pho80Δ</i> and <b>b.</b> the diploid BY4743 <i>pho80Δ</i>, and co-expressed with a second plasmid, containing the yECitrine gene driven by pPho5. Average fluorescence in mid-exponential phase are reported and error bars represent standard deviations of biological triplicates. Increasing expression of <i>gcn5-F221A</i> resulted in decreased mean fluorescence. The data was fit to a Hill-slope competitive inhibition model (dashed line) and IC₅₀ values were extracted (<b>c</b>), indicating the relative promoter strength of <i>gcn5-F221A</i> resulting in half-maximal inhibition. Diploid yeast required nearly twice as strong promoter strength. <b>d.</b> yEcitrine mRNA levels were measured using RT-PCR for select promoter strengths (.07, .16, .32, and .70). The <i>gcn5-F221A</i> mutant serves as a competitive inhibitor to wild-type <i>GCN5</i> acetyltransferase activity.</p>