# Mechanism and reconstitution of circadian transcription in cyanobacteria

## TL;DR

The paper explains how a bacterial circadian clock controls gene activity and demonstrates circadian transcription in a test tube using purified proteins.

## Contribution

The study reveals the mechanism of clock-regulated transcription and reconstitutes it in vitro for sustained circadian gene expression.

## Key findings

- RpaA acts as an activator or repressor of RNA polymerase depending on its binding position.
- A heterologous in vitro system using T7 RNA polymerase sustains circadian transcription for multiple days.
- The findings define minimal components for circadian clock function.

## Abstract

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.

Fang et al. reveal how a bacterial circadian clock turns genes on and off at the right times of day and use the purified proteins to drive circadian gene transcription in a test tube for days.