UBC Theses and Dissertations
GAL4 is regulated by a glucose-responsive functional domain Stone, Geoffrey
Gene regulation in eukaryotes has been a major focus of molecular biological research, aiming to elucidate the genetic processes involved in cell development and disease. To better understand the fundamentals of gene regulation, simple cellular systems such as lower eukaryotes are being studied. These systems are easier to analyze through genetic mutation, providing an overall picture of a particular genetic pathway. One such system is the galactose utilization (GAL) pathway of the yeast Saccharomyces cerevisiae. Expression of the GAL genes is determined by the availability of carbon sources in the media. This regulation is exercised through GAL4, a transcriptional activator of the GAL genes. The availability of galactose induces GAL4 activity by inhibiting the GAL4 negative regulator, GAL80. Glucose, the preferred carbon source, inhibits GAL4 activity by several mechanisms. I demonstrate that one mechanism of glucose repression is mediated by a large, previously uncharacterized, central region of GAL4. This region directly inhibits GAL4 activity in the presence of glucose; deletion of the central region eliminates glucose repression. Fusion of the central region to a heterologous transcriptional activator (LexA-VP16) confers glucose repression. Inhibitory domains in the central region constitutively inhibit activity when a region called the glucose response domain, also present in the central region, is deleted. Inhibition of LexA-VP16 by the central region is accompanied by loss of DNA binding. I suggest that direct inhibition of GAL4 activity in glucose is mediated by interaction of the central region inhibitory domains with the DNA binding and dimerization domain, an interaction which prevents DNA binding. In the absence of glucose, DNA binding is restored by interaction of the glucose response domain with the inhibitory domains.
Item Citations and Data