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

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posted on 27.04.2012, 01:52 by Amanda M. Lanza, John J. Blazeck, Nathan C. Crook, Hal S. Alper

The gcn5-F221A mutant was expressed with varying promoter strengths in a. the haploid BY4741 pho80Δ and b. the diploid BY4743 pho80Δ, 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 gcn5-F221A resulted in decreased mean fluorescence. The data was fit to a Hill-slope competitive inhibition model (dashed line) and IC50 values were extracted (c), indicating the relative promoter strength of gcn5-F221A resulting in half-maximal inhibition. Diploid yeast required nearly twice as strong promoter strength. d. yEcitrine mRNA levels were measured using RT-PCR for select promoter strengths (.07, .16, .32, and .70). The gcn5-F221A mutant serves as a competitive inhibitor to wild-type GCN5 acetyltransferase activity.