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groove. Deletion of this region resulted in a protein able to bind telomere and silencer DNA with high affinity, suggesting that this region is not essential for DNA binding or HTH1-HTH2 cooperative interaction. Deletion of a "loop" region between HTH1 and HTH2 was also performed, and results suggested a role for this region in optimal binding of Rap 1 to tandemly repeated telomere sequences. Rapl is a multifunctional protein in S. cerevisiae. It plays a role in chromatin organization, gene silencing and transcriptional activation. Activation alone takes place using up to 100 Rapl DNA binding sites on the genome. All of these functions are mediated by Rapl binding to the DNA duplex, and the Rapl DBD structure must be adaptive to the requirements of the various cellular processes. This thesis suggests that the HTH1, HTH2, "tail" and "loop" regions are specifically designed to optimize Rapl function. Cooperative binding to DNA by HTH1-HTH2 allows a certain degree of degeneracy at high affinity binding sites. "Tail" interactions, while not essential for DNA binding, maintain binding site specificity at a "core" ACC sequence. Finally, the "loop" region allows proper alignment of HTH1 and HTH2, increasing the cooperative association of these two motifs.

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.