Protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy.

Gene promoters filter transcription factor (TF) localization dynamics and changes in the DNA binding affinity of TFs. Here, we present a protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy. We describe steps for preparing and characterizing a light delivery platform and light-controlled TF mutants. We then detail procedures for subjecting the TFs to light doses that generate defined patterns of localization while measuring fluorescent reporter gene activation via live-cell microscopy. For complete details on the use and execution of this protocol, please refer to Sweeney and McClean..