1997 Fiscal Year Final Research Report Summary
A Novel Cell-Free Peptide Synthesis Driven by Pyridine
| Project/Area Number |
08555200
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
生物・生体工学
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| Research Institution | University of Tokyo |
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Principal Investigator |
WATANABE Kimitsuna University of Tokyo Graduate School of Engineering, Prof., 大学院・工学系研究科, 教授 (00134502)
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| Co-Investigator(Kenkyū-buntansha) |
OOBE Yoshitaka Sumitomo Chemical Co.Ltd.Tsukuba Research Lab.Manager, 筑波研究所, 主席
SHIGA Akinobu Sumitomo Chemical Co.Ltd.Tsukuba Research Lab.Head, 筑波研究所, 所長
NITTA Itaru University of Tokyo Graduate School of Engineering, Assistant Prof., 大学院・工学系研究科, 助手 (30272404)
UEDA Takuya University of Tokyo Graduate School of Engineering, Associate Prof., 大学院・工学系研究科, 助教授 (80184927)
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| Project Period (FY) |
1996 – 1997
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| Keywords | aminoglycoside / in-vitro translation / non-enzymatic tRNA binding / pre-translocational ribosome / puromycin reaction / ribosome-inactivating protein / トランスロケーション / 抗生物質 |
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| Research Abstract |
We have already demonstrated that high concentrations (40-60%) of pyridine was able to promote polypeptide synthesis on ribosomes from various organisms without any protein factors. For better understanding the mechanism of this pyridine system, association of ribosomal subunits, assembly of ribosomal proteins, as well as translocation reaction in 60% pyridine were investigated using E.coli ribosomes. Sucrose density gradient profiles showed that while the subunits once exposed to pyridine failed to re-associate themselves in the aqueous solution even containing 20 mM Mg^<2+>, they could form the total couple in 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins composing the three features of the large subunit, especially those included in the elongation factor-binding domain (the L7/L12 atalk and L11), were released from the reconstituted particle. Both of polyphenylalanine synthesis and translocation activities in 60% pyridine were completely abolished by neomycin, a strong inhibitor against factor-independent translocation, whereas both of the reactions were not affected at all by gypsophilin, a ribotoxin to inhibit factor-dependent translocation. A similar antibiotic inhibitor, thiostrepton, affected neither of the reactions as well, which is consistent with the fact that the antibiotics requires L11 for its binding to the ribosome. These results suggest that translocation actually occurs in the pyridine system, but in a factors-independent (spontaneous) manner.
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