| Co-Investigator(Kenkyū-buntansha) |
YOKOKAWA Takashi Tokyo Institute of Technology, 生命理工学部, 助手 (90242304)
KAWAI Gota University of Tokyo, 工学部, 助手 (70211860)
UEDA Takuya University of Tokyo, 工学部, 助手 (80184927)
NISHIKAWA Kazuya Tokyo Institute of Technology, 生命理工学部, 助教授 (60109262)
SPREMULLI Li ノスカロライナ大学, 理学部, 教授
SPREMULLI Linda lucy University of North Carolina
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| Research Abstract |
The purpose of this project is to investigate the molecular mechanism of the animal mitochondrial (mt) translation system, especially, to elucidate the relationship between genetic code variations [UGA, termination to Trp codons ; AUA, Ile to Met codons ; AGA/AGG, Arg to Ser(invertebrates), Gly(Ascidian) and Termination codons(vertebrates)] found in animal mitochondria and tRNAs complementary to the varied codons, possessing unusual primary and tertiary structures [lack of D loop-T loop interaction ; lack of D arm or T arm]. By joining the knowledge and techniques of our group and Spremulli's group, this project was made possible. [1992] 1) A large-scale preparation of mt tRNAs was made possible by a hybridization method using DNA probes complementary to tRNA sequences. 2)It was elucidated that tRNA^<Ser> complementary to UCN codons possesses an unusual secondary structure. 3)Biologically active ribosomes, initiation factor (IF-2), elongation factors (EF-Tu, EF-G) and aminoacyl-tRNA sy
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nthetasaes were isolated and their properties investigated.
[1993] 1) The recognition sites of tRNA^<Ser> complementary to AGY codons by seryl-tRNA synthetase was investigated. It was elucidated that A44 in the T loop is the most important recognition site. 2) The poly(U)-dependent poly(Phe) synthesis system was constructed for bovine liver mitochondria and the exchangeability of its components with those of E.coli was investigated. 3) A novel modified nucleoside, 5-formylcytidine was found at the first anticodon of bovine mt tRNA^<Met>.4) The higher-order structure of mt tRNA^<Phe> was investigated by the sensitivity with RNase and chemical reagents.
[1994] 1) The poly(U)-dependent poly(Phe) synthesis was activated by addition of 1 mM spermine. 2) To verify that AUA is a Met codon, ribosomal binding and translation of Met-tRNA^<Met> in the presence of AUA-containing mRNA were attempted. 3) By obtaining fMet-tRNA^<Met> with the purified formyltransferase, it was elucidated that fMet-tRNA prefers IF-2 rather than EF-Tu and vice versa for Met-tRNA, indicating that the tRNA^<Met> is bifunctional by formylation of Met-tRNA. Less
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