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

在真核生物基因表达调控中，转录抑制是重要的机制之一。Tup1辅阻遏物是一种转录阻遏物，在多种细胞过程中发挥作用，从酵母到人类都是保守的。尽管出芽酵母、酿酒酵母和白色念珠菌以及裂殖酵母裂殖酵母的Tup1同源物具有转录抑制功能，但它们的转录抑制结构域显示出低序列同源性。本研究利用酵母低温诱导表达系统表达了酿酒酵母Tup1和粟酒裂殖酵母Tup11全长蛋白，通过X射线结构分析和功能分析，探讨了Tup1辅阻遏物的转录抑制机制。酵母宿主菌蛋白酶基因的破坏和降低诱导温度提高了Tup1的表达水平。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200 mg Tup 1蛋白。虽然研究了多种方法来结晶纯化的Tup 1蛋白，但Tup 1蛋白的晶体没有充分生长。白色念珠菌Tup1的组蛋白结合结构域缺失，嵌合Tup1蛋白在链球菌中具有阻遏功能。两个抑制缺陷Tup1突变的组蛋白结合结构域被分离。序列分析表明，其中一个基因的谷氨酸74位被丙氨酸取代，谷氨酰胺118位被精氨酸取代，另一个基因的谷氨酸74位被甘氨酸取代，丝氨酸246位被脯氨酸取代。从酵母细胞中纯化的Tup1和Tup11蛋白被磷酸化。利用大肠杆菌表达了Tup1与Ssn6相互作用域和Ssn6与Tup1相互作用域。coli表达系统。

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.