| Project/Area Number |
07458156
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ANRAKU Yasuhiro The University of Tokyo, Department of Biological Sciences, Graduate School of Science, Professor, 大学院・理学系研究科, 教授 (20012643)
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| Co-Investigator(Kenkyū-buntansha) |
OHYA Yoshikazu The University of Tokyo, Department of Biological Sciences, Graduate School of S, 大学院・理学系研究科, 助教授 (20183767)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | VMA1 Protozyme / Vacuolar membrane ATPase / VDE DNA endonuclease / Protein splicing / WMA1プロトザイム / プロティンスプライシング / VMA1プロトザイム |
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| Research Abstract |
Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions. This unique autocatalytic reaction was first discovered in the yeast VMA1 protozyme where the 50 kDa site-specific endonuclease (VDE) is excised from the 120kDa precursor containing the N- and C-tereminal regions of the catalytic subumit of the vacuolar membrane ATPase. In this work we established a method for measuring in vitro protein splicing as follows : VDEs conjugated with various recombinant polypeptides at both N- and C-terminal ends were expressed in E.coli and examined for their ability to catalyze self splicing. Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies. We demonstrated in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction eff
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iciently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed. Next, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the VMA1 protozyme, and found that these site-specific random mutations interfere with normal protein splicing to different extents. Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal aplicing junction genetically interacts with distal triplet residues preceding the N-terminal junction. This is the first evidence showing that the N-terminal portion of the vacuolar membrane ATPase subunit is involved in protein splicing. Our genetic evidence is consistent with a structural model that correctly aligns two parallel beta-strands ascribed to the triplets. This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions. Less
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