Structural and biochemical characterization of a cyanobacterium circadian clock-modifier protein
Structural and biochemical characterization of a cyanobacterium circadian clock-modifier protein
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DOI：
10.1074/jbc.m608148200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Shimizu, Toshiyuki
中科院分区：
文献类型：
--
作者：
Arita, Kyouhei;Hashimoto, Hiroshi;Shimizu, Toshiyuki
生物钟是自我维持的生化振荡器。蓝藻的振荡器由三个kai基因（kaiA、kaiB和kaiC）的产物组成。KaiC的自磷酸化循环在细胞内以24小时的周期强劲振荡，这对蓝藻生物钟的基本计时至关重要。最近，周期延长基因（pex）被归类为一种与重置相关的基因，其突变体表现出短周期表型。事实上，pex信使核糖核酸（mRNA）和pex蛋白（Pex）在黑暗期增加，并且在昼夜明暗循环条件下，pex突变体的节律相位提前3小时。在此，我们报告了对来自聚球藻Synechococcus elongatus PCC 7942的Pex进行的X射线晶体学分析和生化特性研究。该分子具有带翼螺旋基序的（α + β）结构，且显示以二聚体形式发挥作用。二聚体中的亚基排列独特，在其他带翼螺旋蛋白中未曾见过。使用包含kaiA上游序列的25个碱基对互补寡核苷酸进行的电泳迁移率变动分析表明，Pex对双链DNA具有亲和力。此外，突变分析显示，Pex利用翼区识别DNA。对Pex进行的体内节律分析表明，在缺乏pex基因的聚球藻中，携带无法与DNA结合突变的pex基因的组成型表达不具备周期延长活性，这表明Pex是一种DNA结合转录因子。
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 (alpha+beta) 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.