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Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures
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From primordial clocks to circadian oscillators.

Warintra Pitsawong1,2, Ricardo A P Pádua1, Timothy Grant3,4,5

  • 1Howard Hughes Medical Institute and Department of Biochemistry, Brandeis University, Waltham, MA, USA.

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This study reveals the ancient KaiBC circadian clock mechanism in Rhodobacter sphaeroides, identifying ATP-to-ADP ratio as the key environmental cue driving this primordial biological clock.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chronobiology

Background:

  • Circadian rhythms are fundamental biological processes regulated by post-translational oscillators.
  • The KaiABC system is a well-understood cyanobacterial circadian clock, but ancient KaiBC-only systems remain largely uncharacterized.
  • These KaiBC systems are hypothesized to possess a basic hourglass-like timekeeping mechanism.

Purpose of the Study:

  • To investigate the primordial circadian clock in Rhodobacter sphaeroides, which utilizes only the KaiBC proteins.
  • To elucidate the molecular mechanisms of this ancient timekeeping system in the absence of KaiA.
  • To understand the evolutionary origins of circadian oscillators.

Main Methods:

  • X-ray crystallography and cryogenic electron microscopy were employed to determine the structure of KaiC.
  • Kinetic analysis was performed to identify environmental cues and understand clock dynamics.
  • Structural and kinetic data were integrated to map allosteric networks within the KaiC protein.

Main Results:

  • A novel dodecameric fold for KaiC was discovered, comprising two hexamers linked by a 12-helix coiled-coil bundle.
  • This coiled-coil interaction, mediated by the KaiC carboxy-terminal extension, represents an ancient regulatory element.
  • A long-range allosteric network connecting conformational changes to phosphorylation sites was identified.
  • The ratio of ATP to ADP was identified as the environmental cue that drives the clock's daily cycle.

Conclusions:

  • The KaiBC system in Rhodobacter sphaeroides functions as a primordial circadian oscillator.
  • The identified dodecameric KaiC structure and its allosteric regulation provide insights into the evolution of circadian clocks.
  • Environmental ATP-to-ADP fluctuations are the driving force behind this ancient biological clock, offering a glimpse into early timekeeping mechanisms.