Elucidation of the construction principle of the mitochondria translation system by means of structural biology

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The aim of this research is to elucidate the construction principle of the mitochondria! translation system, by analyzing the functional structures of each component of the animal mitochondrial translation system using X-ray analysis and NMR spectroscopy, focusing on the functional characteristics and structural abnormalities of the components. The following results have been obtained for these three years.(1) Bovine mitochondrial in vitro translation system has been established by optimizing concentration of each component and reaction conditions, which has similar efficiency to that of 」. co// system in terms of poly(U)-dependent poly(Phe) synthesis.(2) Ribosomes are exchangeable between E. coli and mitochondrial systems, as far as tRNA and translation factors of the same origins are used, and EF-Tu of E. coli origin is used. Thus, the ribosomes are functionally equivalent between E. coli and mitochondrial systems.(3) It was verified that 2 species of serine tRNAs with unusual secondary structures can be functional. The low efficiency of the D arm-lacking serine tRNA is derived from a defect in its binding to the ribosomal A site.(4) These two serine tRNAs were recognized by a single seryl-tRNA synthetase (serRS) and its recognition sites on the two tRNAs were identified.(5) A unique EF-Tu was found which recognized almost all the T arm-lacking tRNAs in nematode mitochondria. It had 57 amino acid extension at the C terminus which was considered to complement the T arm-lacking part of tRNAs. Another EF-Tu recognizing two D arm-lacking serine tRNAs was specific for serine.(6) Crystallization of these translation factors are being under way. We have obtained a needle-like srystal for SerRS. The X-ray analysis will be made in future.