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Updated: Jun 27, 2026
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Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter
Published on: September 17, 2016
Functionally important structural elements of the cyanobacterial clock-related protein Pex.
Shunsuke Kurosawa1, Reiko Murakami, Kiyoshi Onai
1Department of Physics, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan.
Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|November 27, 2008
Summary
Pex protein, a key component of the cyanobacterial circadian clock, binds kaiA gene DNA via its wing region. Substitutions in this region disrupt DNA binding, affecting circadian rhythm functions.
Area of Science:
- Molecular Biology
- Chronobiology
- Structural Biology
Background:
- The cyanobacterial circadian clock relies on input pathways involving proteins like Pex.
- Pex protein regulates the expression of the kaiA clock gene, influencing circadian period length.
Purpose of the Study:
- To determine the crystal structure of Anabaena Pex (AnaPex) and Synechococcus Pex (SynPex).
- To identify the DNA-binding sequence and critical regions of Pex involved in kaiA gene regulation.
Main Methods:
- X-ray crystallography to determine Pex protein structure.
- DNase I protection and electrophoresis mobility shift assays to identify Pex-binding DNA sequences.
- Site-directed mutagenesis to assess the role of specific amino acid residues in DNA binding and protein function.
Main Results:
- Pex forms a homodimer with a winged-helix structure.
- A 25-bp imperfect inverted repeat sequence upstream of kaiA was identified as the Pex-binding site.
- Mutations in the Pex wing region abolished DNA binding, while mutations in the alpha3 helix affected protein stability and function.
Conclusions:
- Pex binds to the kaiA upstream DNA sequence through its wing region.
- The alpha3 helix of Pex is crucial for its stability and in vitro function.
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