THE NEUROSPORA CRASSA CIRCADIAN CLOCK: AN ORDERLY LOOP DRIVEN BY A DISORDERED CONDUCTOR

Abstract

Circadian clocks are composed from cell-autonomous molecular oscillators that set gene expression rhythms and thereby enable organisms to pace their metabolism to changes in light and temperature. Transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of the transcriptional repressor proteins, such as the central clock component of the fungal clock, Frequency (FRQ).In the filamentous fungus, Neurospora crassa, the white-collar complex serves as a transcriptional activator that turns on the expression of the frq gene. The resulting protein, Frequency (FRQ) forms a complex with FRH (FRQ-interacting helicase) and Casein-Kinase I (CK1a.) Collectively, the latter three proteins form the FFC complex that then inhibits the white-collar complex activity, thereby resetting the clock. Currently, there is a dearth of structural and biophysical data on FRQ and these complexes. In particular, a key question concerns how FRQ is able to orchestrate the clock with very little predicted tertiary structure. Here I present the reconstitution of FRQ and the FFC complex in varying phosphorylation states and the results of an array of biophysical experiments aimed at defining their structural properties. Our integrative structural biology approach provides the first domain-specific structural details on FRQ and the repressive FFC complex. My experiments demonstrate that FRQ, although mostly disordered, contains an ordered core, undergoes phosphorylation-driven structural changes and uses phase-separation to sequester clock proteins and regulate their enzymatic activities.