The Roles of the Dimeric and Tetrameric Structures of the Clock Protein KaiB in the Generation of Circadian Oscillations in Cyanobacteria
The Roles of the Dimeric and Tetrameric Structures of the Clock Protein KaiB in the Generation of Circadian Oscillations in Cyanobacteria
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DOI：
10.1074/jbc.m112.349092
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发表时间：
2012-08-24
影响因子：
4.8
通讯作者：
Ishiura, Masahiro
中科院分区：
文献类型：
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作者：
Murakami, Reiko;Mutoh, Risa;Ishiura, Masahiro
蓝藻生物钟的分子机制由三种蛋白质KaiA、KaiB和KaiC组成。这三种Kai蛋白相互作用，并在ATP（一种体外KaiABC时钟系统）存在下在体外产生昼夜节律振荡。KaiB由四个亚基组成，组织为二聚体的二聚体，其整体形状是一个细长的六边形板，带正电荷的裂缝两侧是两个带负电荷的脊。我们发现C-末端缺失的突变KaiB（KaiB（1-94））是一个二聚体，它缺少带负电荷的脊.尽管其二聚体结构，KaiB（1-94）与KaiC相互作用，并在体外KaiABC时钟系统中产生正常的昼夜节律振荡。KaiB（1-94）也在蓝藻细胞中产生昼夜节律振荡，但它们很弱，表明KaiB的C-末端区域和四聚体结构对于体内正常基因表达节律的产生是必需的。KaiB（1-94）在我们检测的KaiC结合蛋白中显示出对KaiC的最高亲和力，并抑制KaiC与SasA形成复合物，SasA参与KaiABC时钟振荡器在转录调节中的主要输出途径。这种缺陷解释了表达KaiB的细胞中缺乏正常基因表达节律的机制（1-94）。
The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C- terminal deletion ( KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C- terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94.)