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Mechanism of transcriptional repression of yeast Tup1 corepressor by X-ray structure analysis.

通过 X 射线结构分析酵母 Tup1 辅阻遏物的转录抑制机制。

基本信息

批准号：
17570093
负责人：
MUKAI Yukio
金额：
$ 2.24万
依托单位：
Nagahama Institute of Bio-Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In the control of gene expression in eukaryotes, transcriptional repression is one of the important mechanisms. Tup1 corepressor, a transcriptional repressor in a variety of cellular process, is conserved from yeast to human. Although Tup1 homologs of the budding yeast, Saccharomyces cerevisiae and Candida albicans, and fission yeast Schizosaccharomyces pombe have a function of transcriptional repression, their transcriptional repression domains display low sequence homology. The aim of this study is the investigation of the mechanism of transcriptional repression by Tup1 corepressor in the X-ray structure analysis and functional analysis.The full-length S.cerevisiae Tup1 and S.pombe Tup11 proteins were expressed using a yeast low-temperature-inducible expression system. Protease gene disruptions of yeast host strain and lowering the induction temperature improved Tup1 expression level. About 200 mg of Tup1 protein were obtained from 5 liter of yeast culture in Ni-affinity chromatography, anion exchange chromatography and gel filtration chromatography. Although a variety of methods were examined to crystallize the purified Tup1 protein, the crystals of Tup1 protein did not grow sufficiently. The chimeric Tup1 proteins having the histone-binding domains of C.albicans Tup1, which has a large deletion in histone-binding domain, had a repression function in S.cerevisiae. Two repression-defective Tup1 mutations in the histone-binding domain were isolated. Sequence analysis revealed that one has replacement of glutamate 74 by alanine and glutamine 118 by arginine, the other has replacement of glutamate 74 by glycine and serine 246 by proline. Tup1 and Tup11 proteins purified from yeast cells were phosphorylated. The interaction domain of Tup1 to Ssn6 and that of Ssn6 to Tup1 were expressed using E. coli expression system.

在真核生物基因表达的控制中，转录抑制是重要机制之一。 Tup1 辅阻遏蛋白是多种细胞过程中的转录阻遏蛋白，从酵母到人类都是保守的。尽管芽殖酵母、酿酒酵母和白色念珠菌以及裂殖酵母粟酒裂殖酵母的Tup1同源物具有转录抑制功能，但它们的转录抑制域表现出较低的序列同源性。本研究的目的是通过X射线结构分析和功能分析研究Tup1辅阻遏子的转录抑制机制。利用酵母低温诱导表达系统表达全长酿酒酵母Tup1和S.pombe Tup11蛋白。酵母宿主菌株的蛋白酶基因破坏和降低诱导温度提高了 Tup1 表达水平。通过Ni亲和层析、阴离子交换层析和凝胶过滤层析从5升酵母培养物中获得约200mg的Tup1蛋白。尽管研究了多种方法来使纯化的Tup1蛋白结晶，但Tup1蛋白的晶体没有充分生长。具有白色念珠菌Tup1组蛋白结合域的嵌合Tup1蛋白，其组蛋白结合域有大量缺失，在酿酒酵母中具有抑制功能。组蛋白结合域中分离出两个抑制缺陷型 Tup1 突变。序列分析显示，一种将第74位谷氨酸替换为丙氨酸，将第118位谷氨酰胺替换为精氨酸，另一种将第74位谷氨酸替换为甘氨酸，将第246位丝氨酸替换为脯氨酸。从酵母细胞中纯化的 Tup1 和 Tup11 蛋白被磷酸化。使用大肠杆菌表达系统表达Tup1与Ssn6以及Ssn6与Tup1的相互作用结构域。