ABSTRACT This proposal will be focused on the understanding of mechanisms of two fundamental biological phenomena in eukaryotes: the circadian clock and codon usage bias. Circadian clocks control diverse cellular, physiological, and behavioral processes in eukaryotic organisms. Our long-term goal is to understand the molecular and biochemical mechanisms that permit the measurement of time and the output of circadian rhythms in eukaryotic circadian clocks. Our previous studies made fundamental contributions to the understanding of the eukaryotic circadian clock mechanisms. Synonymous codons are not used with equal frequencies in all genomes examined, a phenomenon called codon usage bias. Even though the phenomenon of codon usage bias has been known for several decades, the functions and mechanisms of codon usage bias are unclear. Our previous work demonstrate that codon usage is a novel layer of the genetic code that can determine both gene expression levels and protein structures. Our lab uses Neurospora, Drosophila and mammalian systems to study these two phenomena. For the circadian clock project, we propose to focus on several key aspects of the circadian oscillator mechanism in both Neurospora and mammalian clock systems. We will determine the role and mechanism of FRQ-CK1a interaction in circadian period determination in Neurospora. In addition, we will expand our study into a mammalian system by determining the role of the PERIOD-CK1 interaction in the mammalian circadian clock. These studies will establish a conserved mechanism for period determination in fungi and animals. Although FRQ in Neurospora and PER proteins in animals are not considered homologous, most of the domains in both proteins are predicted to be intrinsically disordered and both are progressively phosphorylated. We will determine how FRQ and PER function in the circadian clock using biochemical and molecular methods. For the codon usage project, we will build on our ground-breaking discoveries on the roles and mechanisms of codon usage biases in determining gene expression and protein structures. We will determine the mechanism of the codon usage effect on gene transcription in Neurospora based on a previously performed large-scale genetic screen. This study will reveal the mechanisms that underlie the conserved effect of codon usage on gene transcription. We will evaluate how codon usage influences gene expression in mice by creating an in vivo codon usage reporter. This study will establish the mechanism that contributes to effects of codon usage on tissue- and cell type-specific gene expression in mammals. In addition, we will develop a method to modulate translation elongation speed based on the role of codon usage in regulating protein folding that will have potential for use in treatment of many diseases. Together, these studies will address fundamental questions that are critical for our understanding of these two biological phenomena in eukaryotes.