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Principal Investigator: SUSAN S GOLDEN
Organization: UNIVERSITY OF CALIFORNIA, SAN DIEGO
Fiscal Year: 2024
Award: $677,095
Funding agency: National Institute of General Medical Sciences
This project leverages a cyanobacterial model system to answer the following questions: what are the molecular
interactions that mark the passage of time in a cell, where do they occur in the cell, how do they mediate temporal
regulation of events, and why does biological timing matter for fitness? The circadian biological clock is an
oscillatory timer that drives 24-h rhythms of biological activities. Clock dysfunction in humans is related to a
spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and
sleep disorders. However, the circadian clock is pervasive well beyond mammals, promoting fitness in diverse
organisms throughout the phylogenetic tree. 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. 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. A new in vitro preparation comprising the oscillator proteins KaiA, KaiB, and KaiC, along with the
kinases CikA and SasA and the transcription factor RpaA, reconstitutes the circadian rhythm of binding of RpaA
to its target promoter with a real-time readout. This project will apply the in vitro clock and other technical and
conceptual advances towards biochemical, cytological, genomic, and physiological objectives that will answer
the target questions. The in vitro clock will reveal the molecular events that occur when the clock resets to an
environmental timing cue, identify the sites of action of nucleotides that modulate the timing circuit, and determine
how RpaA and a second transcription factor that is regulated by environmental signals, RpaB, work together to
influence circadian phasing. The discovery that the kinases SasA and CikA impart tolerance to fluctuating
oscillator component concentrations will overcome past hurdles for establishing a circadian circuit in Escherichia
coli as a naïve model system for exploring clock connections to cellular physiology and for biotechnology
applications. High-resolution cryo-electron tomography and focused ion-beam milling will be used to visualize
clock-controlled daily changes in intracellular organization and the clock complex itself. The molecular basis and
fitness advantage of circadian control of natural transformation will be determined. A bar-coded transposon
library first used to identify all genes required for photoautotrophic growth will be used to identify new loci that
contribute to fitness in a day-night cycle. Paired with physiological and metabolic assays, these experiments will
answer the question: why does the timing of molecular events matter? Together, these approaches will elucidate
clock mechanisms and the value of the clock to diurnal physiology, and will advance biotechnological
opportunities for controlling metabolism in both photosynthetic and traditional bacterial production systems.
Terms: <Address><Anacystisnidulans><Animals><Assay><Bamboo Grass><Bar Codes><Basal Transcription Factor><Basal transcription factor genes><Binding><Bioassay><Biochemical><Biologic Models><Biological><Biological Assay><Biological Clocks><Biological Models><Biological Rhythm><Biotech><Biotechnology><Blue-Green Algae><Blue-Green Bacteria><Cancers><Cardiovascular Diseases><Cell Body><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Circadian Dysregulation><Circadian Rhythms><Clock protein><Complex><Cryo-electron tomography><Cues><Cyanobacterium><Cyanophyceae><Cyanophyta><Cytology><Disease><Disorder><Dysfunction><E coli><E. coli><Environment><Escherichia coli><Eukaryota><Eukaryote><Event><Functional disorder><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Genes><Genetic><Genomics><Growth><Health><Human><In Vitro><Intermediary Metabolism><Intracellular Communication and Signaling><Ions><Kinases><Libraries><Malignant Neoplasms><Malignant Tumor><Mammalia><Mammals><Mediating><Mental disorders><Mental health disorders><Metabolic><Metabolic Processes><Metabolic syndrome><Metabolism><Model System><Modeling><Modern Man><Molecular><Molecular Interaction><Nucleotides><Nyctohemeral Rhythm><Organism><Personal Satisfaction><Phase><Phenotype><Phosphotransferase Gene><Phosphotransferases><Phylogenetic Analysis><Phylogenetics><Physiologic><Physiological><Physiology><Physiopathology><Preparation><Production><Prokaryotae><Prokaryotic Cells><Property><Protein Biochemistry><Protein/Amino Acid Biochemistry><Proteins><Psychiatric Disease><Psychiatric Disorder><Regulation><Resolution><S elongatus><S. elongatus><Sasa><Signal Transduction><Signal Transduction Systems><Signaling><Site><Sleep Disorders><Subcellular Process><Synechococcus elongatus><System><Time><Tissue Growth><Transcription Factor Proto-Oncogene><Transcription factor genes><Transphosphorylases><Trees><Twenty-Four Hour Rhythm><Visualization><Work><barcode><biologic><biological signal transduction><body clock><cardiovascular disorder><circadian><circadian abnormality><circadian clock><circadian disruption><circadian disturbance><circadian dysfunction><circadian impairment><circadian pacemaker><circadian process><cryo-EM tomography><cryoEM tomography><cryoelectron tomography><daily biorhythm><electron cryo-tomography><experiment><experimental research><experimental study><experiments><fitness><internal clock><living system><malignancy><mental illness><model organism><neoplasm/cancer><ontogeny><pathogen><pathophysiology><preparations><prokaryote><promoter><promotor><psychiatric illness><psychological disorder><reconstitute><reconstitution><resolutions><sleep diseases><sleep dysfunction><sleep illness><sleep problem><transcription factor><well-being><wellbeing>