Characterization Of Transcriptional Activators In The Fungal Circadian Clock

Abstract

Circadian clocks are a common biological feature that allows organisms to anticipate changes in their environments by synchronizing their biological activity to the Earth's day and night cycle. These clocks are transcriptional/translational feedback loops, where transcriptional activators act as positive elements to promote the expression of negative elements, which in turn inhibit their own formation. While significant progress has been made in elucidating the function of circadian clock proteins, relatively little is known about their interactions on the molecular level. To address this issue, I investigated the transcriptional activators White Collar (WC) I and II, which in the well characterized clock model N. crassa associate to form the White Collar Complex (WCC) and promote the expression of the negative clock element Frequency (FRQ). Also studied were the related WC homologs from Phycomyces blakesleeanus, Trichoderma atroviride, and Cryptococcus neoformans. Bioinformatic analysis of these proteins was used to design protein fragments based on variations in their domain architecture. Once generated, expression and purification conditions were optimized allowing for the generation of soluble variants, for crystallization screening and biophysical characterization using small-angle X-ray scattering. Results from these studies indicate the presence of a PAS AB repeat in both N. crassa WC I and II. Purification of the PAS domain containing proteins with and without the proteins C-terminal zinc fingers resulted in apparent dimers in size-exclusion chromatography (SEC) experiments. Copurification of WC I and II proteins resulted in the formation of a tetrameric PAS mediated complex as shown by SDS-PAGE, SEC, light-scattering and small-angle X-ray scattering, suggesting the formation of a PAS mediated WCC containing four zinc fingers, which may facilitate the binding of multiple DNA sequences in the FRQ promoter region during transcription. ii