This project addresses two fundamental questions: how does a circadian clock function at the molecular level as a timekeeping mechanism, and how is it integrated at the cellular level to control activities such as gene expression and cell division? The circadian clock is an oscillatory timer that drives 24-h rhythms of biological activities in diverse organisms from bacteria to humans, and disruptions in its underlying molecular mechanism adversely affect fitness. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. Despite different strategies for timekeeping that have evolved between cyanobacteria and mammals, the circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. A quantitative, systems- level, biochemical understanding is attainable for the circadian clock of S. elongatus, whose fundamental circadian oscillator can be reconstituted in vitro with three proteins, KaiA, KaiB, and KaiC. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. Moreover, as is true in mammalian cells, the circadian clock of S. elongatus controls the timing of cell division. This project will leverage recent conceptual and technical advances to determine the mechanism of the timekeeping system with unprecedented clarity, understand how the clock controls activities in the cell, and elucidate how a sense of time is inherited when cells divide. The discoveries that KaiB refolds as part of the timekeeping mechanism and becomes a connector between oscillator and output pathways, and that metabolites are sampled by oscillator proteins to set the clock with local time, will enable establishment of a more complete in vitro clock that exhibits rhythmic output relevant for control of gene expression. Using this preparation, and kinetics measurements of partner interactions from BioLayer Interferometry, the project will quantify the steps that contribute to timekeeping, synchronization, and rhythmic output. Analysis in vivo of mutations that alter such interactions will tie specific steps to clock functions. The biochemical basis of interactions between two transcription factors that integrate temporal and environmental cues will be clarified. Time-lapse measurements of dividing cells that carry fluorescently labeled clock proteins and markers of the circadian cycle, in genetic backgrounds that are proficient or deficient in clock-control of cell division, will provide insight into how clock components are inherited with the correct timestamps. Proteomic approaches will identify partners responsible for localization of clock components within the cell and the ability of the clock to allow or disalow cytokinesis. Together, these approaches will elucidate clock mechanisms and the relationship between the circadian and cell division cycles.

Public Health Relevance

The project uses a simple cyanobacterial model system to ask questions that cannot be addressed as directly in animals about how the circadian biological clock keeps time, stays synchronized with the environment, and controls cell division. Circadian dysfunction contributes to a range of disease states in humans, from metabolic syndrome to mental illness. Past discoveries using this system have revealed fundamentally new paradigms of protein biochemistry with relevance for diverse organisms, including humans and pathogens.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1-TRN-7 (MR))
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Sesma, Michael A
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University of California San Diego
Schools of Arts and Sciences
La Jolla
United States
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Tseng, Roger; Goularte, Nicolette F; Chavan, Archana et al. (2017) Structural basis of the day-night transition in a bacterial circadian clock. Science 355:1174-1180
Hughes, Michael E; Abruzzi, Katherine C; Allada, Ravi et al. (2017) Guidelines for Genome-Scale Analysis of Biological Rhythms. J Biol Rhythms 32:380-393
Boyd, Joseph S; Cheng, Ryan R; Paddock, Mark L et al. (2016) A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock. J Bacteriol 198:2439-47