Defining necessary circadian clock elements The circadian clock in organisms as diverse as fungi and humans have a rather similar structure: Timing depends on daily cycles of transcription in circuits in which feedback loops control the timing of oscillations. A critical role has been ascribed to negative elements, which lead to inhibition of their own transcription, and to degradation of these elements, which is signaled by phosphorylation events. However, Larrando et al. show that in the fungus Neurospora , after manipulations that prevent phosphorylation-signaled degradation of the negative element FREQUENCY (FRQ), rhythms still persist (see the Perspective by Kramer). They suggest a model in which other phosphorylation events on Frq (of which there are over 100) must have critical roles in controlling the clock, independent of negative element degradation. Science , this issue 10.1126/science.1257277 ; see also p. 476 Control of negative circadian elements in the simple fungus Neurospora goes beyond targeted proteolysis. Also see Perspective by Kramer INTRODUCTION Circadian oscillators allow individual organisms to coordinate metabolism with day/night cycles and to anticipate such changes. Such oscillators in fungi and animals share a common regulatory architecture centered on transcription and translation-based negative feedback loops. Within such oscillators, extensive coordinated and progressive phosphorylation of negative element proteins leads to their proteasome-mediated degradation. Current clock models posit that this turnover event is the final essential step in the loop and that the time taken to achieve phosphorylation and turnover determines the speed of the circadian clock. The clock in Neurospora exemplifies such oscillators: FREQUENCY (FRQ) is a negative element, and its half-life is well correlated with circadian period length. Surprisingly, however, using real-time reporters in cells with compromised proteasomal turnover, we unveiled an unexpected uncoupling between negative element half-life and circadian period determination. RATIONALE We followed FRQ dynamics as well as transcriptional activity of the frq promoter in vivo using luciferase-based reporters. FRQ turnover was tracked through Western blotting, and kinase inhibitors helped to test the correlation between phosphorylation and period length. Strains bearing frq alleles causing abnormal period lengths were used, as were strains with diminished FRQ turnover, including knockouts of both the F-box protein FWD-1 (a ubiquitin ligase that mediates FRQ proteasomal degradation) and individual components of the COP9 signalosome. RESULTS Without FWD-1, FRQ turnover is severely compromised and circadian regulation of development is lost; however, in such Δ fwd-1 cells, the amount of FRQ still oscillated, the result of cyclic transcription of frq and reinitiation of FRQ synthesis. The circadian nature of these rhythms was confirmed by examining well-established frq mutants having altered periods. Analyses of additional strains bearing knockouts of individual COP9 signalosome components further confirmed circadian oscillations in FRQ amounts, despite compromised FRQ turnover. Broadly accepted oscillator models posit that negative element stability determines clock period length; thus, Δ fwd-1 strains with long FRQ half-lives are predicted to have extremely long periods. This, however, is not seen: Period is mainly determined by the characteristics of the frq allele irrespective of the half-life of this negative element. Partial inhibition of overall phosphorylation provided additional evidence that clock protein phosphorylation events, not the resulting stability changes, provide key information in determining period length. DISCUSSION The long-standing and assumed causal loop uniting clock protein phosphorylation, stability, and period determination should be revisited. Data indicate that qualities of FRQ—in particular, its phosphorylation status rather than its quantity—are crucial for determining when the circadian feedback loop is completed and can be restarted. Previously described strong correlations between clock protein phosphorylation and half-life and between half-life and period length are, in fact, just correlations that do not always imply cause and effect. Although degradation is the final outcome of FRQ posttranslational modifications, phosphorylation and its effects of secondary, tertiary, and quaternary protein structure may actually be the key elements determining clock speed. Although it may be premature to broadly generalize these findings to all circadian oscillators, diverse data from several animal circadian systems are not inconsistent with this revised model. Distinct roles for FRQ phosphorylation and degradation in the clock. White Collar-1 and -2 (WC-1 and WC-2) activate frq expression and FRQ (with FRH and CK1) later inhibit expression. FRQ phosphorylation affects interactions with WC-1/WC-2, reducing inhibition. By influencing these key interactions, FRQ phosphorylations determine the rate at which core clock events, those within the clock face, occur. After key phosphorylations close the loop, degradation-related events need not affect circadian period. 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. 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. Unexpectedly, we unveiled normal circadian oscillations that reflect the allelic state of frq but that persist in the absence of typical degradation of FRQ. This manifest uncoupling of negative element turnover from circadian period length determination is not consistent with the consensus eukaryotic circadian model.