Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators
Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators
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DOI：
10.1105/tpc.002733
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发表时间：
2002-09-01
期刊：
影响因子：
11.6
通讯作者：
Yamazaki, T
中科院分区：
文献类型：
--
作者：
Hosoda, K;Imamura, A;Yamazaki, T
B基序是拟南芥中参与组氨酸-天冬氨酸磷酸化信号转导系统的B型反应调节因子（ARR）的特征。同源模体广泛存在于植物转录因子GARP家族中。为了获得对B基序（或GARP基序）的结构和功能的一般了解，我们表征了来自代表性ARR ARR 10的B基序，这导致了许多有趣的发现。首先，ARR 10的B基序（命名为ARR 10-B，从Thr-179延伸到Ser-242）具有核定位信号，如绿色荧光蛋白-ARR 10-B融合蛋白在洋葱表皮细胞中的细胞内定位所示。第二，纯化的ARR 10-B分子在体外特异性结合具有核心序列AGATT的DNA。这是证明了几个在体外的方法，包括PCR辅助的DNA结合位点的选择，凝胶阻滞试验，和表面等离子体共振分析。最后，通过核磁共振光谱确定了ARR 10-B在溶液中的三维结构，表明其含有螺旋-转角-螺旋结构。此外，ARR 10-B和靶DNA之间的相互作用模式通过NMR光谱进行了广泛的评估。总之，这些结果使我们提出，ARR 10-B的DNA识别机制基本上是相同的同源结构域。我们的结论是，B基序是一个多功能的结构域负责核定位和DNA结合，并建议这些见解可以普遍适用于大GARP家族的植物转录因子。
The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.