Phase‐dependent generation and transmission of time information by the KaiABC circadian clock oscillator through SasA‐KaiC interaction in cyanobacteria

Abstract

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation‐enhancing activity of KaiC, ultimately resulting in genome‐wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 has the domain structure of an orthodox histidine kinase and that its C‐terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature‐ and phosphorylation state‐dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N‐terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation‐enhancing activity of KaiC requires the C‐terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.

The Roles of the Dimeric and Tetrameric Structures of the Clock Protein KaiB in the Generation of Circadian Oscillations in Cyanobacteria

“…Therefore, it is not likely that SasA and KaiB bind to the same site on KaiC. We have demonstrated by surface plasmon resonance analysis and gel filtration chromatography that the SasA binding site of KaiC is located on its N-terminal domain (21), as suggested by NMR analysis (35). The SasA inhibition of KaiB WT -KaiC WT complex formation has been demonstrated by Native-PAGE using proteins derived from Synechococcus (36).…”

“…A series of these reactions and interactions among Kai proteins generate circadian oscillations such as oscillations in the phosphorylation level (4) and ATPase activity (5) of KaiC and complex formation among Kai proteins (6,7). SasA also associates preferentially with p-KaiC, which enhances the autophosphorylation of SasA (21). KaiB and SasA compete each other for KaiC (Fig.…”

“…S2). Therefore, it is likely that KaiB regulates the normal SasA-KaiC interaction required for the transmission of time information from KaiC to the transcription apparatus, resulting in genome-wide transcription cycles in cyanobacterial cells (21,32,33). SasA trimer and KaiC hexamer associate each other at a 1:1 molar ratio at their N-terminal domains, and the phosphorylation states of their C-terminal domains affect the affinity of the interaction (21).…”

“…We estimated protein concentrations using the Bio-Rad Protein Assay with BSA as the standard as previously described (20). Unless otherwise stated, KaiA, KaiB, KaiC, and SasA refer to the KaiA dimer, KaiB tetramer, KaiC hexamer, and SasA trimer (21), respectively, and their concentrations are expressed in terms of their oligomeric status.…”

An allele of the crm gene blocks cyanobacterial circadian rhythms

“…Structural changes corresponding to ordered cycling of the phosphorylation state of KaiC are crucial for interactions with clock output effectors (18,19). Disruption of rpaA has been reported to result in attenuated expression from the kaiBC promoter and to abolish cyclic phosphorylation of the core oscillator protein KaiC, which appears to be constitutively phosphorylated in an rpaA null strain under constant light (LL) conditions (10).…”