| Project/Area Number |
17570093
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Nagahama Institute of Bio-Science and Technology |
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Principal Investigator |
MUKAI Yukio Nagahama Institute of Bio-Science and Technology, Bioscience, Associate Professor, バイオサイエンス学部, 准教授 (60252615)
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| Co-Investigator(Kenkyū-buntansha) |
OHGIYA Satoru National Institute of Advanced Industrial Science and Technology, Research Institute of Genome-based Biofactory, Deputy Director, ゲノムファクトリー研究部門, 副研究部門長 (30356620)
MATSUMURA Hiroyoshi Osaka University, Graduate School of Engineering, Assistant Professor, 大学院工学研究科, 助教 (30324809)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | yeast / transcriptional repression / Tup1 / low-temperature-inducible / corepressor / X-ray structure expression system / 遺伝子発現調節 / X線結晶構造解析 |
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| Research Abstract |
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.
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