Codon usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1–3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4–6. Many genes exhibit little codon usage bias, which is thought to reflect a lack of selection for mRNA translation. Alternatively, however, non-optimal codon usage may have biological significance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ level but surprisingly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. In this study, we show that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a L strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt L mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ∼32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a L strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a L strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka RIP strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka RIP strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop. In Neurospora, Drosophila, and mammals, the positive elements are all heterodimeric complexes, consisting of two PER-ARNT-SIM (PAS) domain-containing transcriptional factors that bind to the cis-elements in the promoter of the negative elements to activate their transcription. On the other hand, the negative elements repress their own transcription by inhibiting the activity of the positive elements through their physical interactions. It is unclear how negative elements inhibit the activity of positive elements to close the circadian negative feedback loops. Since the identification of the Drosophila doubletime (dbt) gene, which encodes for a casein kinase I (CKI) homolog, it has become clear that post-translational protein phosphorylation is essential for the function of circadian clocks Price et al. 1998). Despite the evolutionary distance, remarkable conservation of post-translational regulation exists among different eukaryotic systems from fungi to human (see Discussion;Liu 2005;Heintzen and Liu 2006).In the filamentous fungus Neurospora crassa, the core circadian negative feedback loop consists of four essen-

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events. mass spectrometry ͉ casein kinase ͉ frequency E ukaryotic circadian oscillators from fungi to mammals are controlled by autoregulatory negative feedback loops (1-4). In the filamentous fungus Neurospora crassa, 2 protein complexes function in the core circadian negative feedback loop (5, 6). WHITE COLLAR complex (WCC), formed by WC-1 and WC-2, activates transcription of the frequency ( frq) gene by binding to its promoter (7-13). On the other hand, FFC (consisting of FRQ and the FRQ-interacting RNA helicase, FRH) inhibits WCC activity by promoting the phosphorylation, and consequently repression, of frq transcription (12,(14)(15)(16)(17)(18).Like the animal PERIOD (PER) proteins, FRQ is progressively phosphorylated after its synthesis and becomes extensively phosphorylated before its disappearance, resulting in a robust oscillation of its phosphorylation profile (19). One role of FRQ phosphorylation is to promote FRQ degradation through the ubiquitin-proteasome pathway mediated by ubiquitin E3 ligase SCF FWD-1 . FWD-1 acts as the substrate-recruiting subunit that recognizes and binds phosphorylated FRQ (20)(21)(22). Under normal conditions, FRQ is phosphorylated by CKII, and PKA (12,16,19,[23][24][25]. In the ck-1a (casein kinase 1a), cka (catalytic subunit of CKII), and ckb-1 (regulatory subunit of CKII) mutants, FRQ is hypophosphorylated and more stable relative to the wild type, resulting in arrhythmia or long-period phenotypes (12,23,25). These results suggest that CK-1a and CKII phosphorylate and promote FRQ degradation. In contrast, PKA counters the role of casein kinases by stabilizing FRQ (12,16). FRQ is also dephosphorylated and stabilized by protein phosphatases PP1 and PP4 (17,2...

Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. On the other hand, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. Here, by using whole-cell isotope labeling and quantitative mass spectrometry methods, we show that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. We further show that the cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop.[Keywords: Circadian clock; Neurospora; protein kinase A; phosphorylation; casein kinase I] Supplemental material is available at http://www.genesdev.org. Cheng et al. 2001aCheng et al. , 2005. FFC represses the transcription of frq by inhibiting WCC activity through their physical interaction (Aronson et al. 1994;Merrow et al. 1997Merrow et al. , 2001Cheng et al. 2001aCheng et al. , 2003Denault et al. 2001;Froehlich et al. 2003;He et al. 2006). This circadian negative feedback loop generates the robust circadian rhythms of frq RNA and FRQ protein in constant darkness (DD) .Post-translational modification of clock proteins by phosphorylation plays essential roles in all circadian clocks (Price et al. 1998;Lowrey et al. 2000;Lin et al. 2002;Sathyanarayanan et al. 2004

SummaryNickel serves as a cofactor for various microbial enzymes including superoxide dismutase (SOD) found in Streptomyces spp. In Streptomyces coelicolor , nickel represses and induces production of Fecontaining and Ni-containing SODs, respectively, primarily at the transcriptional level. We identified the nickel-responsive regulator (Nur), a Fur (ferric-uptake regulator) homologue, which binds to the promoter region of the sodF gene encoding FeSOD in the presence of nickel. Disruption of the nur gene caused constitutive expression of FeSOD and no induction of NiSOD in the presence of nickel. The intracellular level of nickel was higher in a D nur mutant than in the wild type, suggesting that Nur also regulates nickel uptake in S. coelicolor . A putative nickel-transporter gene cluster ( nikABCDE ) was identified in the genome database. Its transcription was negatively regulated by Nur in the presence of nickel. Purified Nur protein bound to the nikA promoter region in a nickeldependent way. These results support the action of Nur as a regulator of nickel homeostasis and antioxidative response in S. coelicolor , and add a novel nickel-responsive member to the list of versatile metal-specific regulators of the Fur family.