Reconstitution of an intact clock reveals mechanisms of circadian timekeeping

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire...

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Bibliographic Details
Published in:	Science (American Association for the Advancement of Science) Vol. 374; no. 6564; p. eabd4453
Main Authors:	Chavan, Archana G, Swan, Jeffrey A, Heisler, Joel, Sancar, Cigdem, Ernst, Dustin C, Fang, Mingxu, Palacios, Joseph G, Spangler, Rebecca K, Bagshaw, Clive R, Tripathi, Sarvind, Crosby, Priya, Golden, Susan S, Partch, Carrie L, LiWang, Andy
Format:	Journal Article
Language:	English
Published:	United States The American Association for the Advancement of Science 08-10-2021
Subjects:	Adenosine triphosphate ATP Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Binding Biological clocks Biology Cellular communication Circadian rhythm Circadian Rhythm - genetics Circadian Rhythm - physiology Circadian Rhythm Signaling Peptides and Proteins - chemistry Circadian Rhythm Signaling Peptides and Proteins - genetics Circadian Rhythm Signaling Peptides and Proteins - metabolism Circadian rhythms CLOCK protein Clock systems Complexity Constraining Control theory Cooperativity Coupling (molecular) Crystal defects Crystallography Cyanobacteria Deoxyribonucleic acid DNA Electrophoresis Feedback (Response) Feedback loops Fluorescence Fluorescence polarization Gel electrophoresis Gene expression Gene Expression Regulation, Bacterial Gene regulation Histidine Histidine kinase In vivo methods and tests Kinases Macromolecules Mathematical analysis Mathematical models Metabolism Mimicry Molecular Mimicry Mutagenesis Mutants Mutation Phenotypes Phosphorylation Phosphotransferases - chemistry Phosphotransferases - genetics Phosphotransferases - metabolism Polyacrylamide Promoter Regions, Genetic Protein Domains Protein Folding Protein interaction Protein Kinases - metabolism Protein Multimerization Protein turnover Proteins Representations Signal transduction Sunset Synechococcus - genetics Synechococcus - metabolism Synechococcus - physiology Synthetic biology Transcription factors Transcription, Genetic
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Summary:	Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author contributions: Conceptualization: A.G.C., J.A.S., J.H., S.S.G., C. L.P., and A.L. Methodology: A.G.C., J.H., J.A.S., C.S., D.C.E., M.F., and C.R.B. Investigation: A.G.C., J.A.S., J.H., C.S., D.C.E., M.F., J.G.P., R.K.S., and S.T. Validation: A.G.C., J.A.S., J.H., C.S., D.C.E., M.F., and S.T. Formal analysis: A.G.C., J.H., J.A.S., C.R.B., P.C., C.S., D. C.E., and M.F. Resources: S.S.G., C.L.P., and A.L. Data curation: A.G.C., J.H., J.A.S., and C.S. Writing – original draft: A.G.C., J.H., J.A.S., C.L.P., and A.L. Writing – review and editing: A.G.C., J.A.S., J.H., D.C.E., C.R.B., S.S.G., C.L.P., and A.L. Funding acquisition: J.H., J.G.P., P.C., S.S.G., C.L.P., and A.L. Supervision: S.S.G., C.L.P., and A.L. These authors contributed equally to this work.
ISSN:	0036-8075 1095-9203
DOI:	10.1126/science.abd4453