Coiled-coil domain-mediated FRQ–FRQ interaction is essential for its circadian clock function in Neurospora

The frequency (frq) gene, the central component of the frq-based circadian negative feedback loop, regulates various aspects of the circadian clock in Neurospora. However, the biochemical function of its protein products, FRQ, is poorly understood. In this study, we demonstrated that the most conserved region of FRQ forms a coiled-coil domain. FRQ interacts with itself in vivo, and the deletion of the coiled-coil region results in loss of the interaction. Point mutations, which are designed to disrupt the coiled-coil structure, weaken or completely abolish the FRQ self-association and lead to the arrhythmicity of the overt rhythm. Mutations of the FRQ coiled-coil that inhibit self-association also prevent its interaction with two other key components of the Neurospora circadian clock, namely WC-1 and WC-2, the two PAS domain-containing transcription factors. Taken together, these data strongly suggest that the formation of the FRQ–FRQ and FRQ–WC complexes is essential for the function of the Neurospora clock.