Study on tRNA recognition mechanism by aminoacyl-tRNA synthetase from archaea

虽然tRNA识别元件在包括大肠杆菌在内的真细菌系统中得到了广泛的研究，但对于被归类为第三界的古细菌中氨基酰基-tRNA合成酶（ARS）的tRNA识别位点知之甚少。为了研究极端嗜热古细菌Aeropyrum pernix K1对每一种ARS识别苏氨酸、脯氨酸和色氨酸tRNA的分子机制，克隆并过表达了每一种ARS基因。利用体外野生和突变tRNA转录本和过表达ars进行氨基酰化实验。在苏氨酸系统中，结果支持了我们之前的发现，即A.pernix ThrRs不识别苏氨酸tRNA的鉴别碱基。这一结果表明，古细菌中苏氨酸tRNA的鉴别碱基的重要性存在差异，因为嗜盐古细菌中苏氨酸tRNA的鉴别碱基是同源ThrRS的重要决定因素。结果还发现，尽管存在n端截断的独特结构，但A.pernix ThrRs识别苏氨酸tRNA的受体茎区和其他物种的ThrRs一样多。此外，在G36U移植的同时，第二对和第三对受体茎突变为C-G的脯氨酸tRNA的体外转录被a.p ennix ThrRS苏氨酸化。这种仅移植5个碱基就从脯氨酸tRNA转变为苏氨酸tRNA的结果表明，冬氨酸苏氨酸tRNA的主要身份元件是受体茎区和反密码子。在脯氨酸系统中，鉴定碱基A73、G1-C72碱基对以及反密码子G35和G36是a.p ennix pros的强决定因子。G1-C72碱基对的识别以及反密码子G35和G36是a.p enix pro的重要决定因素。对G1-C72碱基对的识别与大肠杆菌ProRS系统有较大差异。结果表明，在色氨酸系统中，抗密码子C34、C35和A36、鉴别碱基G73、G1-C62和g2 - c71碱基对受体茎端的trpr特异性识别。这些结果表明，三个物种的ARS对tRNA的识别机制是不同的。少

Although the tRNA identity elements have been widely investigated in eubacterial system including Escherichia coli, little is known about tRNA recognition sites for aminoacyl-tRNA synthetase (ARS) from archaea, which is classified as a third kingdom. To study the molecular mechanism of recognition of threonine, proline and tryptophan tRNA for each ARS from extreme thermophilic archaeon, Aeropyrum pernix K1, each ARS gene was cloned and overexpressed. Aminoacylation experiments using in vitro wild and mutant tRNA transcripts and overexpressed ARSs were examined. In the threonine system, the results support our previous finding that A.pernix ThrRs does not recognize the discriminator base of threonine tRNA. This result indicated the diversity of importance of the discriminator base among archaea because the discriminator base of threonine tRNA from halophilic archaeon, Haloferax volcanii, is a strong determinant by cognate ThrRS. It was also found that A.pernix ThrRs did recognize the ac … More ceptor stem region of threonine tRNA as well as other species ThrRS, in spite of N-terminal truncated unique structure. Furthermore, it was shown that in vitro transcript of proline tRNA whose second and third pairs of acceptor stem was mutated to C-G in addition to transplantation of G36U was threonylated by A.pernix ThrRS. This result of switching from proline tRNA to threonine tRNA by transplanting only five bases indicates that the major identity elements of A.pernix threonine tRNA are acceptor stem region and anticodon. In the proline system, it was found that the discriminator base A73, G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRs. Recognition of G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRS. Recognition of G1-C72 base pair was quite different from E.coli ProRS system. In the tryptophan system, results showed that anticodon C34, C35 and A36, discriminator base G73, G1-C62 and G2-C71base pairs of acceptor stem end were base specifically recognized by A.pernix TrpRS. These results indicate that the tRNA recognition mechanisms by ARS is different among three kingdomes. Less