| Co-Investigator(Kenkyū-buntansha) |
NITTA Itoru The University of Tokyo, Graduate School of Engineering, Assistant Prof., 大学院・工学系研究科, 助手 (30272404)
KAWAI Gota Chiba Institute of Technology, Faculty of Engineering, Assistant Prof., 工学部, 講師 (70211860)
UEDA Takuya The University of Tokyo, Graduate School Frontier Science, Associate Prof., 大学院・新領域創成科学研究科, 助教授 (80184927)
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| Research Abstract |
This research aims to elucidate the molecular mechanism of animal mitochondrial (mt) translation system through carrying out (1) structural analysis of animal mt tRNAs, and (2) verification of translation ability of these tRNAs by using a mitochondrial in vitro translation system. The animal mt translation system possesses the distinct features as compared with those of prokaryotic and eukaryotic cytoplasmic systems ; exsistences of tRNA possessing unusual secondary structures and non-universal genetic code, only 22 species of tRNAs exist in mitochondria, a single tRNA^<Met> is used for both initiator and elongator, ribosomes contain rRNA with shorter chain lengths and much more proteins. We have isolated and characterized individual factors involved in the translation system and got the following new findings. 1) We found several non-universal genetic codes in various animal nit systems and analyzed the primary structures of tRNAs decoding the codes. it turned out that the genetic code variations are mainly induced by the base (mainly modified base) in the anticodon first position (Wobble position), which lead to the establishment of the mt specific Wobble rule. 2) By combining biochemical experiments and ^1H-NMR analyses we verified that even tRNAs possessing trancated secondary structures lacking either D arm or T arm could be folded into L-shape-like tertiary structures. 3) We succeeded to construct an in vitro translation system composed of only mt translation apparatuses. By using this system we proved that AUA isoleucine codon is translated as methionine by tRNA^<Met> having a modified base, f^6C at the anticodon first position. 4) Aminoacyl-tRNA synthetase, elongation factors, release factors, and formyltransferase were purified from bovine mitochondria, characterized, and their expression system was established in E.coli. We analyzed molecular mechanism of interaction between these factors and tRNA.
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