Structural and biochemical characterization of circadian clock related protein Pex in Synechococcus sp. Strain PCC 7942

Graduate School of Engineering, kyoto University* Yokohama city university/International Graduate School of Arts and Sciences**
â—‹Kyouhei Arita* Kumiko Igari** Mayuko Akaboshi** Toshiyuki Simizu** Hiroshi Hashimoto** Toshiyuki Shimizu** Shinsuke Kutsuna** Mamoru Sato**

Circadian clocks are self-sustained biochemical oscillators. The oscillator of Synechococcus elongates is constituted by the products of the Kai genes (KaiA, KaiB, and KaiC). KaiC auto-phosphorylation cycle robustly oscillates in the cell with 24 hr period and is essential for basic timing process of the cyanobacterial circadian clock. Recently, period extender (pex), whose deficient mutant shows short period phenotype, was also isolated as a resetting-related gene. In fact, pex mRNA and pex protein (Pex) increase in dark period, and the pex mutant subjected diurnal light-dark cycles showed phase-advance of the rhythm by 3 h. Furthermore, disruption of pex gene in cyanobacteria cell leads to greatly enhancement of KaiA gene activity. To more fully understand the molecular mechanism involved in regulation of circadian clock oscillation in cyanobacteria, we have determined the crystal structure of Pex in Synechococcus sp. Strain PCC 7942 at 1.8 Å resolution. Pex has a winged-helix motif in the mid portion similar to that of several DNA binding proteins. Pex dimerizes in a unique fashion compared with other winged-helix DNA binding proteins. Electrophoretic Mobility Shift Assay of Pex with DNA containing KaiA promoter sequence demonstrates that Pex has DNA binding activity and specifically recognize KaiA promoter sequence. Mutation analysis shows that Pex uses the winged-helix region to recognize DNA. Our structural and biochemical data suggest Pex potentially regulates KaiA expression levels by interaction with the KaiA promoter region as a negative transcriptional regulator.