Characterization of the N-terminal domain of the yeast transcriptional repressor Tup1 -: Proposal for an association model of the repressor complex Tup1•Ssn6
Characterization of the N-terminal domain of the yeast transcriptional repressor Tup1 -: Proposal for an association model of the repressor complex Tup1•Ssn6
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DOI：
10.1074/jbc.275.12.9011
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发表时间：
2000-03-24
影响因子：
4.8
通讯作者：
Wolberger, C
中科院分区：
文献类型：
--
作者：
Jabet, C;Sprague, ER;Wolberger, C
酵母Tup1和Ssn6蛋白形成一个转录抑制复合体，抑制大量基因的转录。研究表明，Tup1蛋白的n端结构域与Ssn6蛋白的一个区域相互作用，该区域由一个四肽基序的10个串联拷贝组成。在这项工作中，我们使用表面等离子体共振分析来测量Tup1的n端结构域对酿酒酵母SsnG最小3-TPR结构域的亲和力，该结构域足以与Tup1结合。SsnG的这个结构域与酿酒酵母和白色念珠菌Tup1的结合具有相当的亲和力，但与含有点突变的Tup1蛋白的亲和力低100倍，该蛋白在体内会导致抑制缺陷。利用分析性超离心、CD光谱、有限蛋白水解和H-1核磁共振的研究结果表明，Tup 1的这个结构域主要是α -螺旋结构，并形成一个高度非小叶形状的稳定四聚体。从Tup1四聚域的无序晶体中记录的x射线衍射包含典型的卷曲线圈的纤维衍射。我们的研究结果被用来提出Tup1的n端结构域及其与SsnG蛋白相互作用的模型。
The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae SsnG that is sufficient for binding to Tup1. This domain of SsnG binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and H-1 NMR show that this domain of Tup 1 is primarily alpha-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the SsnG protein.