|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
To investigate the recognition and discrimination sites of E.coli several tRANs for each aminoacyl-tRNA synthetase (ARS), various unmodified mutant transcripts were prepared by in vitro transcription system with T7 RNA polymerase. In the case of tRNA^<Thr>, G_<35> and U_<36> of anticodon and two base pairs of acceptor stem, G_1-C_<72> and C_2-G_<71>, were the recognition sites by ThrRS.Discriminator base, A73, was not involved in the charging activity. Other class I tRNAs, including aspartic acid, arginine, lysine and proline tRNAs, were recognized both sites of anticodon and discriminator base by each cognate ARS.Interesting recognition sites were determined such as A_<20> in variable pocket of tRNA^<Arg>, modified U_<34> in the anticodon of tRNA^<Lsy>, G_<37> in anticodon loop, G_<49> in TPSI stem, G_<72> in the acceptor stem and U_<17A> in the D-loop of tRNA^<Pro>, respectively. Recognition and discrimination sites of classII tRNAs having a long variable arm, tRNA^<Leu> and tRNA^<Ser>, were also examined. The discriminator base A_<73> and A_<14> in D-loop, but not the anticodon, were found to serve as critical recognition elements of tRNA^<Leu>.In case of tRNA^<Ser>, all of the characteristic nucleotide of tRNA^<Ser> were not involved in recognition by SerRS.The SerRS selectively recognizes tRNA^<Ser> on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA^<Ser>.