| Project/Area Number |
18570106
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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 | Nara Institute of Science and Technology |
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Principal Investigator |
KOJIMA Chojiro Nara Institute of Science and Technology, Graduate School of Biological Sciences, Associate Professor (50333563)
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| Co-Investigator(Kenkyū-buntansha) |
MISHIMA Masaki Tokyo Metropolitan University, Graduate School of Science and Engineering, Associate Professor (70346310)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,020,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Structural Biology / NMR / Signal Transduction / Protein / Molecular Recognition / 耕造生物学 |
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| Research Abstract |
IRE1 on endoplasmic reticulum (ER) is a transmembrane protein (type I) with N- and C-terminal domains in lumen and cytoplasm sides, respectively, and the N-terminal domain senses the accumulation of denatured proteins. Activated Irelp specifically cleaves mRNA of transcription activation factor HAC1 of the endoplasmic reticulum chaperon genes, and enables translation of Haclp by performing splicing without spliceosome. This new splicing has characteristics such as (1) 5' and 3' sides are cleaved independently, (2) tRNA ligase is involved, (3) it is performed in cytoplasm, and it is the signal transduction mechanism which is widely conserved from yeast to human. Here, we focused on the elucidation of the structural bases of IRE1 pathway in ER stress response, in particular the splicing mechanism without spliceosome.
The structural studies of Ire1p RNase domain, C-terminal domain and the stem loop of HAC1 mRNA including cleavage site are performed by NMR and crystallization. The major results are the following. (1) For NMR experiments, the Irelp RNase domain is enriched with ^13C and ^15N, and the sample conditions including buffers and temperature are optimized. The best HSQC spectrum is obtained with weak alkaline HEPES buffer. (2) The expression and purification of the Irelp C-terminal domain are successfully performed, and subjected to crystallization with the aid of Professor Toshio Hakoshima (NAIST) . The crystals are obtained but they are not suitable for structure analysis. (3) Two stem loops of HAC1 mRNA including cleavage site are used for the structural study using NMR. Imino proton signals indicate the presence of the base pair not only in stem regions but also in loop regions. (4) The variants of functionally important residues are prepared and used to study the RNase activities and the structural changes. (5) NMR titration experiments are performed to identify the interface residues between the Irelp RNase domain and the stem loop of HAC1 mRNA.
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