2006 Fiscal Year Final Research Report Summary
Development of a new translation system for animal proteins: the engineering strategy of ribosome function
| Project/Area Number |
16310139
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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 |
Applied genomics
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| Research Institution | Niigata University |
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Principal Investigator |
UCHIUMI Toshio Niigata University, Institute of Science and Technology, Professor, 自然科学系, 教授 (50143764)
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| Co-Investigator(Kenkyū-buntansha) |
NOMURA Takaomi Shinsyu University, Faculty of Textile Science Technology, Assistant, 繊維学部, 助手 (90362110)
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| Project Period (FY) |
2004 – 2006
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| Keywords | ribosome / protein synthesis / GTPase-associated center / L12 / IRES / L11 / PO / P1 / P2 |
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| Research Abstract |
In this project, we analyzed functional structures in the translational machinery and modified them by molecular engineering in order to develop a new translation system useful for synthesis of eukaryotic proteins. The following are major results of this project.
1. The functional structure of protein complex located in the ribosomal GTPase-associated center.
In eukaryotes, a couple of P1-P2 heterodimers bound to the C-terminal regions of PO and formed a pentameric complex. In eubacteria, 2〜3 L12 homodimers (differing with species) bound to the C-terminus of L10. In archaebacteria, 3 L12 homodimers bound to PO and formed a heptameric complex, as revealed by biochemical analysis and X-ray crystallography. By removing one or two dimers from each the complex, the activity of GTPase and polypeptide synthesis was reduced.
2. Mechanism of translation initiation mediated with the IRES of an insect virus (PSIV).
We elucidated that 1) the IRES binds to the ribosome through interaction of the small subunit protein S25, 2) induces some conformational changes in the ribosome, 3) activates the ribosomal accessibility to elongation factor eEF2, and then 4) enhances binding of the first aminoacyl-tRNA to the ribosomal P site. We also succeeded translation of luciferase mediated by the IRES using an IRES-luciferase fusion mRNA. By using this system, we clarified that the 3'-untranlated region of the PSIV IRES-carrying gene participates in the translation efficiency.
3. Development of a new in vivo translation system.
The E.coli mutant strain dL11 lacks L11 ribosomal protein that is a component of the GTPase-associated center. This strain showed very low growing rate (114〜115 of wild type) and was used for a host cell of protein synthesis. We demonstrated that this E. coli strain is useful for synthesis of eukatyotic proteins that cannot be obtained as a soluble state by usual strains.
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