RECOGNITION MECHANISM OF tRNA FOR AMINOACYL-tRNA SYNTHETASE

为了研究几种反式大肠杆菌对每种氨基酰-tRNA合成酶(ARS)的识别和识别位点，利用T7RNA聚合酶体外转录系统制备了各种未修饰的突变体转录本。对于tRNA^&lt；Thr&gt；，反密码子的G_&lt；35&gt；和U_&lt；36&gt；，以及受体茎的两个碱基对G_1-C_&lt；72&gt；和C_2-G_&lt；71&gt；是ThrRS的识别位点。鉴别碱基A73不参与充电活动。另一类tRNAs，包括天冬氨酸、精氨酸、赖氨酸和脯氨酸tRNAs，在反密码子和识别碱基上都被同源ARs识别。在tRNA^；lt；Arg&gt；的可变口袋中确定了感兴趣的识别位点，在tRNA^；lt；lt；Lsy&gt；，G&lt；37&gt；中确定了感兴趣的识别位点，如tRNA^；；lt；Lsy&gt；，G；；分别在tRNA^&lt；Pro&gt；的D-环中。还研究了具有长可变臂的第II类tRNAs的识别和识别位点，tRNA^；lt；Leu&gt；和tRNA^；发现D-loop中的鉴别碱基A_&lt；73&gt；和A_&lt；14&gt；是tRNA^；lt；Leu&gt；的关键识别元件。对于tRNA^；lt；Ser&gt；，tRNA^；；Ser&gt；的所有特征核苷酸都不参与SerRS的识别。SerRS根据tRNA^；；Ser&gt；的特征核苷酸选择性地识别tRNA^；；

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>.