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

在真核生物的基因表达调控中，转录抑制是重要的机制之一。Tup1辅阻遏子是一种在多种细胞过程中的转录抑制因子，在酵母和人类之间都是保守的。虽然芽生酵母、酿酒酵母、白色念珠菌和裂殖酵母的Tup1同源物具有转录抑制功能，但它们的转录抑制域表现出低序列同源性。本研究的目的是在X射线结构分析和功能分析中探讨Tup1辅阻遏子转录抑制的机制。酵母菌蛋白酶基因的破坏和诱导温度的降低提高了Tup1的表达水平。经镍亲和层析、阴离子交换层析和凝胶过滤层析，从5L酵母培养物中可获得约200 mg的Tup1蛋白。虽然对纯化的Tup1蛋白进行了多种结晶方法的研究，但Tup1蛋白的晶体生长不够充分。具有白念珠菌Tup1组蛋白结合区的嵌合蛋白在酿酒酵母中具有抑制功能，在组蛋白结合区有较大的缺失。在组蛋白结合区分离到两个抑制缺陷的Tup1突变。序列分析表明，一个用丙氨酸代替谷氨酸74，用精氨酸代替谷氨酰胺118，用甘氨酸代替谷氨酸74，用脯氨酸代替丝氨酸246。从酵母细胞中纯化的Tup1和Tup11蛋白被磷酸化。利用大肠杆菌表达系统表达了Tup1与SSN6的相互作用结构域以及SSN6与Tup1的相互作用结构域。

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.