| Project/Area Number |
12306005
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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 | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
MASAKI Haruhiko The University of Tokyo, Graduate School of Agricultural and Sciences, Professor (50134515)
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| Co-Investigator(Kenkyū-buntansha) |
HIDAKA Makoto The University of Tokyo, Graduate School of Agricultural and Life Sciences, Associate Professor (50183918)
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| Project Period (FY) |
2000 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥46,650,000 (Direct Cost: ¥40,500,000、Indirect Cost: ¥6,150,000)
Fiscal Year 2003: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000)
Fiscal Year 2002: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
Fiscal Year 2001: ¥12,740,000 (Direct Cost: ¥9,800,000、Indirect Cost: ¥2,940,000)
Fiscal Year 2000: ¥20,000,000 (Direct Cost: ¥20,000,000)
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| Keywords | colicin / tRNA / ribonuclease / tRNase / restriction enzyme / Escherichia coli O157 / molecular mimicry / anticodon / 大腸菌 / 反応機構 / O157 / コドン / インヒビター / 塩基触媒 / RNA制限酵素 / ピオシン / バクテリオファージ / 酸塩基触媒 |
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| Research Abstract |
We characterized colicins E5 and D as "cytotoxic tRNases", a new type of toxins that specifically cleave tRNA anticodon loops. On the infection in sensitive Escherichia coli, the C-terminal ribonuclease domain of colicin E5, E5-CRD, enters the cell and cleaves anticodons of tRNA^<Tyr>, tRNA^<His>, tRNA^<Asn> and tRNA^<Asp> to kill the cell. Likewise, the C-terminal domain of colicin D, D-CRD, cleaves the 3' terminal of anticodon loops of four is accepting tRNA^<Arg> molecules.
The X-ray crystallographic study and mutation experiments revealed that E5-CRD specifically recognizes the common GU dinucleotide in susceptible tRNA anticodons, by mimicking the structure and function of corresponding codons of mRNA. Thus, E5-CRD can be said as an "RNA restriction enzyme" that cleaves single stranded GU sequences. On the other hand, the inhibitor protein of colicin E5, ImmE5, which originally protects the colicinogenic cells from lethality by the cognate colicin, was shown to bind E5-CRD at the substrate site by mimicking tRNA anticodons. This double molecular mimicry represents a novel process that a pair of proteins has acquired a specific protein-protein interaction by taking over an RNA-RNA interaction through an RNA-protein interaction. We also determined the structures of D-CRD and the D-CRD/ImmD complex, though we failed to visualize interacting structures of D-CRD and the substrate. Since tRNA anticodon loop structures are conserved among a wide range of organisms, E5-CRD and D-CRD function even in eukaryotic cells and are expected to be novel tools to analyze eukaryotic cell mechanisms.
We also screened a collection of E. coli 0157 strains for new type tRNase colicins other than E5 and D. We could not discover such a colicin but found that about 1/4 of the whole O157 collection proved to produce some colicins and that unusually more than 3/4 of those colicins were colicin D.
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