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Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.

ABSTRACT: In the cyanobacterium Synechococcus elongatus (PCC 7942) the proteins KaiA, KaiB, and KaiC are required for circadian clock function. We deduced a circadian clock function for KaiA from a combination of biochemical and structural data. Both KaiA and its isolated carboxyl-terminal domain (KaiA180C) stimulated KaiC autophosphorylation and facilitated attenuation of KaiC autophosphorylation by KaiB. An amino-terminal domain (KaiA135N) had no function in the autophosphorylation assay. NMR structure determination showed that KaiA135N is a pseudo-receiver domain. We propose that this pseudo-receiver is a timing input-device that regulates KaiA stimulation of KaiC autophosphorylation, which in turn is essential for circadian timekeeping.

SUBMITTER: Williams SB

PROVIDER: S-EPMC137721 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.

Williams Stanly B SB Vakonakis Ioannis I Golden Susan S SS LiWang Andy C AC

Proceedings of the National Academy of Sciences of the United States of America 20021115 24

In the cyanobacterium Synechococcus elongatus (PCC 7942) the proteins KaiA, KaiB, and KaiC are required for circadian clock function. We deduced a circadian clock function for KaiA from a combination of biochemical and structural data. Both KaiA and its isolated carboxyl-terminal domain (KaiA180C) stimulated KaiC autophosphorylation and facilitated attenuation of KaiC autophosphorylation by KaiB. An amino-terminal domain (KaiA135N) had no function in the autophosphorylation assay. NMR structure ...[more]

PMID: 12438647

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Roles for ClpXP in regulating the circadian clock in <i>Synechococcus elongatus</i>.

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Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.

Project description:In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.

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Damped circadian oscillation in the absence of KaiA in Synechococcus.

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NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: implications for KaiA-KaiC interaction.

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Project description:The core oscillator of the circadian clock of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. The KaiABC oscillator can be re-constituted in vitro with the purified proteins in buffer containing ATP and Mg2+. The interaction between KaiA and KaiC has not been well studied. In this article, different KaiA mutants were designed and used to elucidate the influence of KaiA structure on its function in the in vitro system. Molecular dynamics simulations were adopted to study the structural flexibility of KaiA homodimer. The data presented in this article provide further experimental supports to our work in Chen et al. (2017) [1].

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Circadian transcriptional profile of Synechococcus under the kaiA overexpression

2013-12-31 | GSE47015 | GEO

Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus.

Project description:Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.

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Structure, function, and mechanism of the core circadian clock in cyanobacteria.

Project description:Circadian rhythms enable cells and organisms to coordinate their physiology with the cyclic environmental changes that come as a result of Earth's light/dark cycles. Cyanobacteria make use of a post-translational oscillator to maintain circadian rhythms, and this elegant system has become an important model for circadian timekeeping mechanisms. Composed of three proteins, the KaiABC system undergoes an oscillatory biochemical cycle that provides timing cues to achieve a 24-h molecular clock. Together with the input/output proteins SasA, CikA, and RpaA, these six gene products account for the timekeeping, entrainment, and output signaling functions in cyanobacterial circadian rhythms. This Minireview summarizes the current structural, functional and mechanistic insights into the cyanobacterial circadian clock.

| S-EPMC5892564 | biostudies-other

Circadian transcriptional profile of Synechococcus under the kaiA overexpression

Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock in continuous light (LL) conditions. Here, we employed high-density oligonucleotide arrays to investigate comprehensive profiles of genome-wide Synechococcus gene expression in kaiA-overexpressor (Ptrc::[GTG]kaiA) strains under LL. KaiC has been proposed to globally activate gene expression, our analysis revealed that dawn expressing genes were downregulated by kaiA-overexpression, such that the clock was arrested at subjective dawn. IPTG-inducible kaiA-overexpressor (oxA) S. elongatus PCC 7942 strains were analyzed under continuous light (LL) using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing predicted 2,515 protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also to the almost homologous strain, S. elongatus PCC 7942: a single experiment in oxA under LL in the presence or absence of an inducer, IPTG, from hour 24 to 48 in LL timecourse data (24~48 hours under continuous light) from Synechococcus elongatus PCC 7942 (inducible kaiA-overexpressor) strains

2013-12-31 | E-GEOD-47015 | biostudies-arrayexpress

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