Molecular genetic studies on kai gene : mechanism of circadian oscillator

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Since we have started the current project for molecular basis of cyanobacterial circadian oscillator in 1999, the progress of research has been very fruitful and we obtained following results. Parts of these results have been published in Proc. NAS (2000), Cell (2000), Science (2000) and highly appreciated by colleagues.1) ATP/GTP binding of KaiC and circadian rhythmsKaiC protein carries two ATP/GTP binding motifs. We confirmed that KaiC bound to ATP and introduction of mutations to the motifs completely nullified the ATP binding and the circadian rhythms, suggesting this activity is crucial for the oscillation.2) Circadian rhythms in Kai protein level and KaiC phosphorylationProtein level of three Kai proteins showed circadian rhythms with phases delayed by 8 h from mRNA rhythms. KaiC protein were phosphorylated and the level of phosphorylation also showed circadian rhythms.3) SasA, a histidin kinase that interact with RaiC.A sensory histidine kinase that bound with KaiC was cloned. Disruption of SasA lowered KaiC expression and amplitude of the rhythm. These results indicate that SasA function as amplifier of the circadian oscillator by promoting KaiC expression.4) KaiA enhance phosphorylation of KaiCKaiA protein promotes phosphorylation of KaiC both in vivo and in vitro. This results suggest the mechanism of kaiBC gene activation by RaiC.5) A phytochrome-like protein CikA was cloned with collaboration with Golden's labo (Texas A&M). Disruption of CikA lowered phase shift by 5 h dark pulse and suggest that CikA communicate light signal to the oscillator to reset phase.