Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora

May 23, 2023The Journal of biological chemistry

Parts of the central negative protein FRQ needed to interact with its activator WCC in Neurospora’s core circadian clock

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Abstract

Multiple regions on FREQUENCY (FRQ) are necessary for its interaction with the White Collar complex (WCC).

The interaction between the FRQ-FRH complex (FFC) and WCC is essential for repressing FRQ expression.
High salt conditions can disrupt the physical interaction between FFC and WCC but not within the individual complexes.
Three clusters of negatively charged residues on FRQ are critical for the formation of the FFC-WCC complex.
Mutations that reduce FFC-WCC interaction do not affect the oscillation period of the circadian clock, suggesting that the interaction is necessary for clock function but not for period length.

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In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.

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Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora

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