Circadian rhythms in the cyanobacterium Synechococcus elongatus share the properties of those driven by eukaryotic clocks: an endogenous free-running period of about 24 h, resetting of the phasing of the rhythms by environmental cues, and temperature compensation of circadian period. However, the cyanobacterial circadian oscillator proteins, KaiA, KaiB, and KaiC, are not homologous to those of eukaryotes, and the fundamental clock mechanism is different. A circadian oscillation in phosphorylation of KaiC can be reconstituted in vitro with only the three Kai proteins and ATP. Thus, the mechanism of circadian oscillation has become tractable to biophysical analyses. The current proposal will complement in vitro data by revealing mechanisms that integrate circadian oscillation with fundamental cellular processes. Molecular structures of the oscillator proteins, and the S. elongatus genome sequence, are known. Prior results showed that sigma factors of RNA polymerase affect circadian gene expression, and inactivation of one, SigC, revealed two different periods of oscillation in cells. We created mutations in 68% of all S. elongatus genes and identified new loci that affect circadian period and tie the clock to protein metabolism and redox regulation. A temporally-controlled modification of KaiA was detected in association with a redox sensor, LdpA. An effective fluorescent reporter for real-time intracellular localization of clock proteins was developed. Project 1 will capitalize on these data to provide a comprehensive understanding of a circadian clock at a systems level. Our new Specific Aims are to: (1) determine the connection between redox sensing and clock components by identifying the modification on the subpopulation of KaiA that co-purifies with LdpA, and testing the hypothesis that redox state affects circadian period by changing the ratios of Kai proteins; (2) define unknown components of the periodosome (clock complex) that tie the oscillator to input and output pathways by using ion trap electrospray mass spectrometry of peptides from affinity-tagged complexes; and (3) track the intracellular localization of clock proteins during the circadian and cell cycles and determine whether clock proteins associate with the bacterial nucleoid. Lay summary: This project will explain how circadian oscillations are tied to fundamental processes in living cells, and how the clock can be manipulated. Lessons can be extrapolated to human health and disease, such as metabolic syndrome.
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