![]() Consistent with this, mutant strains in which ubiquitin-dependent proteasomal degradation of FRQ is severely altered show neither overt rhythms nor molecular circadian rhythmicity in FRQ abundance, as detected by Western blotting ( 15, 19, 25, 26). That FRQ stability is a determinant of period length has been supported by the fact that strains bearing long-period frq alleles display a more stable FRQ, whereas this protein exhibits decreased half-life in strains with shorter periods ( 9, 14, 17, 18). FWD-1 is the ortholog of mammalian β-TrCP ( 22) and Drosophila Slimb ( 23, 24), which have similar roles in those circadian systems. A key aspect in FRQ degradation is the association of its hyperphosphorylated isoforms with FWD-1, the substrate-recruiting subunit of an SCF (SKP/Cullin/ F-box) –type ubiquitin ligase that facilitates FRQ ubiquitination and presentation to the proteasome ( 17– 21). More specifically, data supporting the importance of phosphorylation dynamics to clock protein turnover and period determination come from animals ( 10) and Neurospora ( 14, 17, 18) in which precise spatiotemporal arrangement of phosphorylation events dictates period and controls negative element stability. Such changes, including timely turnover, are thought to underlie the daily rhythms in negative element abundance and phosphorylation that characterize eukaryotic circadian oscillators ( 13, 16). Beginning shortly after its synthesis, FREQUENCY (FRQ) is progressively phosphorylated, finally reaching a multi-phosphorylated (hyperphosphorylated) state that leads to its proteasome-mediated degradation ( 14, 15). ![]() In the case of Neurospora crassa, transcription of the frq gene encoding the negative element FRQ is controlled by the White-Collar complex (WCC, the positive element), a heterodimer of PAS-containing GATA transcription factors White Collar-1 (WC-1) and White Collar-2 (WC-2). Thus, in the circadian paradigm for fungi and animals, (Per-Arnt-Sim) domain –containing transcription factors drive the expression of genes whose products lead to the inhibition of their own transcription only after degradation of these products can reinitiation of the cycle begin, and period length is thus obligately coupled to turnover kinetics of clock proteins ( 9– 13). Although posttranslational oscillators have been described ( 7, 8), their generalizability is still being tested. In eukaryotes, the circadian oscillator underlying these subcellular clocks is generally viewed as comprising transcription and translation-based negative feedback loops (TTFLs) with interconnected feedback loops ( 1). In organisms as diverse as humans, mice, fungi, insects, cyanobacteria, and plants, the molecular components of the circadian clocks have been identified in molecular detail ( 1, 3– 6). This manifest uncoupling of negative element turnover from circadian period length determination is not consistent with the consensus eukaryotic circadian model.Ĭircadian clocks provide individuals with the ability to anticipate daily changes associated with the transition of day to night ( 1, 2). Unexpectedly, we unveiled normal circadian oscillations that reflect the allelic state of frq but that persist in the absence of typical degradation of FRQ. The Neurospora crassa circadian negative element FREQUENCY (FRQ) exemplifies such proteins it is progressively phosphorylated at more than 100 sites, and strains bearing alleles of frq with anomalous phosphorylation display abnormal stability of FRQ that is well correlated with altered periods or apparent arrhythmicity. The mechanistic basis of eukaryotic circadian oscillators in model systems as diverse as Neurospora, Drosophila, and mammalian cells is thought to be a transcription-and-translation –based negative feedback loop, wherein progressive and controlled phosphorylation of one or more negative elements ultimately elicits their own proteasome-mediated degradation, thereby releasing negative feedback and determining circadian period length.
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