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J. Biol. Chem., Vol. 282, Issue 2, 1128-1135, January 12, 2007

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Structural and Biochemical Characterization of a Cyanobacterium Circadian Clock-modifier Protein^*

Kyouhei Arita¹, Hiroshi Hashimoto, Kumiko Igari, Mayuko Akaboshi, Shinsuke Kutsuna, Mamoru Sato, and Toshiyuki Shimizu²

From the International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 and the Graduate School of Arts and Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan

Circadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an ( + ) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor.

Received for publication, August 24, 2006 , and in revised form, November 9, 2006.

The atomic coordinates and structure factors (code 2E1N) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Scientific Research on Priority Areas Grant-in-Aids 17054035 (to T. S.), 17048023 (to H. H.), and 18054026 (to M. S.), a national project on protein structural and functional analyses (Protein 3000 project) (to T. S., H. H., and M. S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grant-in-Aid 18770091 for Young Scientists (B) (to H. H.) from the Japan Society of the Promotion of Science (JSPS), and a Kaneko-Narita grant from the Protein Research Foundation (to H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.

² To whom correspondence should be addressed. Tel.: 81-45-508-7226; Fax: 81-45-508-7365; E-mail: shimizu{at}tsurumi.yokohama-cu.ac.jp.

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