The Yeast Transcription Factor GAL4: A Model for Understanding Eukaryotic Transcription
O'Neal, Melissa Ann
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The 26S proteasome regulates numerous cellular pathways, including transcription, through proteolytic and non-proteolytic methods. The Kodadek and Johnston laboratories recently established a novel function for the proteasomal ATPases: the destabilization of activator-DNA complexes. This is independent of proteolysis but requires direct activator-ATPase interactions as well as ATP hydrolysis. The Gal4 mutant Gap71, which is hyper-sensitive to destabilization from a GAL promoter, was instrumental to this discovery. Gal4, but not Gap71, was mono-ubiquitylated in a HeLa nuclear extract and in vivo, suggesting that mono-ubiquitylation of an activator is critical to resisting destabilization by the proteasomal ATPases. To gain a better understanding of these events, the three amino acid substitutions in the Gap71 DNA-binding domain were individually cloned and analyzed for their contributions to the function of Gal4. The data showed that Serine 22 and Lysine 23 but not Lysine 25 were important for the efficiency of the activator. The charge at Lysine 23 was found to be important for Gal4-based transcription and subsequent in vitro work revealed that Gal4 was not only phosphorylated at Serine 22 but that this phosphorylation event was essential for the function of the activator. Many times a phosphorylation event precedes a mono-ubiquitylation event on an activator. Knowing the kinase and ligase machinery that modifies Gal4 would permit us to further test our model. As a result, I designed selection screens in an attempt to isolate the kinase and/or ligase machinery components that modify Gal4. While these particular enzymes were not identified, other novel genes were found to negatively affect the galactose utilization pathway, MSU1 and SPS1. Altogether, the data demonstrated that two post-translation events, phosphorylation and mono-ubiquitylation, prevent an activator-DNA complex from being disrupted, leading to an elegant model in which the proteasomal ATPases act as an important check point in transcription.