INVESTIGATING THE STRUCTURAL PROPERTIES OF ACTIVATORS AND REPRESSORS IN EUKARYOTIC CIRCADIAN CLOCK

Many organisms have an internal circadian clock that helps them adapt to the external changes in light and temperature. The clock is important for cell cycle regulation and genome integrity. Disrupting the internal circadian clock can cause depression, obesity, cardiovascular disease, and even cancer. Importantly, circadian clocks are highly conserved among eukaryotes. Thus, we decided to focus on the well-characterized model systems- fungus Neurospora crassa and fly Drosophila melanogaster to study the structural properties of the interacting circadian clock components. These clock components make up the positive and negative arms of the circadian oscillators. The Neurospora crassa circadian clock consists of a positive regulator White Collar Complex (WCC) that activates the transcription of clock gene frequency. Frequency (FRQ) acts as a repressor in the negative arm of the oscillator. FRQ acts as a repressor by binding with its partner Casein Kinase 1 (CK1) to phosphorylate and inactivate WCCs. Similar to FRQ, the Drosophila circadian clock is composed of a repressor Period (PER) that phosphorylates and inactivates Drosophila positive activators. While many biochemical and genetic studies have explored the functional aspects of the circadian clock components, very little is known about the mechanisms of the interactions with each other. Therefore, an analogous study of the components in both model systems would provide better understanding of the respective protein structures and interactions. In this thesis, we show that the N. crassa WCC can recognize and tightly bind to frq promoter regions in the absence of WC1 photosensor domain. Secondly, N. crassa repressor FRQ is stabilized and phosphorylated if co-purified with binding partner CK1. Additionally, FRQ:CK1 has a more flexible conformation when it is hyperphosphorylated. Lastly, like FRQ, repressor PER in D. melanogaster, is stabilized by phosphorylation on specific sites. This shows structural similarities between these two repressors FRQ and PER. These results provide a deeper understanding of the molecular bases of the circadian clocks in N. crassa and D. melanogaster to pave way for future structural studies of many eukaryotic clock proteins