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<p>Circadian biological clocks evolved across kingdoms of life as an adaptation to predictable cycles of sunrise and sunset. In the cyanobacterium <i>Synechococcus</i> <i>elongatus</i>, a protein-based clock precisely controls when different genes are turned on and off during the 24-h day but the phasing mechanism remains unclear. Here we show the molecular basis of this regulation and reconstitute clock-controlled transcription in vitro using purified components. Biochemical and structural analyses revealed that the clock-regulated transcription factor RpaA can function as either an activator or a repressor of cyanobacterial RNA polymerase, depending on its binding position relative to core promoter elements. Leveraging the repressor mechanism, we developed a heterologous in vitro system driven by bacteriophage T7 RNA polymerase that sustains circadian transcription for multiple days. These findings explain how a single clock output generates opposite phases of gene expression and define the minimal components for circadian clock function, enabling synthetic or biotechnological applications.</p>

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Mechanism and reconstitution of circadian transcription in cyanobacteria

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月10日

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  • Mingxu Fang,
  • Yajie Gu,
  • Miron Leanca,
  • Mariusz Matyszewski,
  • Andy LiWang,
  • Yulia Yuzenkova,
  • Kevin D. Corbett,
  • Susan S. Golden

摘要

Circadian biological clocks evolved across kingdoms of life as an adaptation to predictable cycles of sunrise and sunset. In the cyanobacterium Synechococcus elongatus, a protein-based clock precisely controls when different genes are turned on and off during the 24-h day but the phasing mechanism remains unclear. Here we show the molecular basis of this regulation and reconstitute clock-controlled transcription in vitro using purified components. Biochemical and structural analyses revealed that the clock-regulated transcription factor RpaA can function as either an activator or a repressor of cyanobacterial RNA polymerase, depending on its binding position relative to core promoter elements. Leveraging the repressor mechanism, we developed a heterologous in vitro system driven by bacteriophage T7 RNA polymerase that sustains circadian transcription for multiple days. These findings explain how a single clock output generates opposite phases of gene expression and define the minimal components for circadian clock function, enabling synthetic or biotechnological applications.