(92e) A "Cellular Oscilloscope" to Measure the Frequency Response of Gene Expression

The kinetics of gene expression has been widely studied in response to step changes in signal. Frequency response studies represent a complementary approach to elucidating the kinetics of reaction systems involved in gene expression. We have developed a ?cellular oscilloscope? in S. cerevisiae to measure the frequency response of gene expression ?output? with respect to an oscillating ?input? transcription factor concentration. This system relies on the fact that many eukaryotic transcription factors continuously shuttle between nucleus and cytoplasm and are regulated vis-à-vis their nuclear localization. We employ the phosphate responsive transcription factor Pho4p and show it can be forced in and out of the nucleus quickly (5-10 minutes) and reliably if one eliminates endogenous feedback control in the phosphate-sensing pathway. Microfluidic devices are employed to drive the Pho4p oscillations by varying the phosphate concentration. Using fluorescent proteins, we can measure both the oscillations in Pho4p localization and its effect on downstream gene expression in real-time. Using this system, we study how the promoter architecture of several Pho4p target promoters influences gene expression. Specifically, we ask whether a change in the nuclear concentration (thermodynamic affinity model) or in the nuclear residence time (kinetic proofreading model) of the transcription factor is critical for activation. Finally, we demonstrate the modularity of our system by isolating the minimal shuttling domain of Pho4p and fusing it to alternate DNA binding and activation domains. This will enable the study of promoters that are not part of the endogenous phosphate starvation pathway.