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

虽然在包括大肠杆菌在内的真细菌系统中已经对tRNA识别元件进行了广泛的研究，但对于被归类为第三个王国的古细菌氨基酰-tRNA合成酶(ARS)的tRNA识别位点却知之甚少。为了研究极端嗜热古生物Pernix Aeropyrum Pernix K1每个ARS基因识别苏氨酸、脯氨酸和色氨酸tRNA的分子机制，我们克隆并高效表达了每个ARS基因。用体外野生型和突变型tRNA转录本和过表达的ARS进行了氨基酰化实验。在苏氨酸系统中，结果支持我们之前的发现，即Pernix ThrRs不识别苏氨酸tRNA的识别碱基。这一结果表明，鉴别碱基在古菌中的重要性是不同的，因为嗜盐古细菌苏氨酸tRNA的鉴别碱基是同源ThrRS的一个很强的决定因素。还发现A.pernix ThrRs确实识别Ac…更多的苏氨酸tRNA以及其他物种的受体茎区域ThrRs，尽管N-末端截短独特的结构。此外，还发现，在G36U移植的基础上，第二和第三对受体茎突变为C-G的Proline tRNA的体外转录本被A.pernix ThrRS苏氨酸化。这一结果表明，Pernix苏氨酸tRNA的主要识别元件是受体、茎区和反密码子。在Pro系统中，识别碱基A73、G1-C72碱基对、反密码子G35和G36是Pernix ProRs的强决定因素。G1-C72碱基对的识别以及反密码子G35和G36是Pernix ProRS的强决定因素。对G1-C72碱基对的识别与E.ColiProRS系统有很大的不同。结果表明，在色氨酸系统中，反义密码子C34、C35和A36、识别碱基G73、G1-C62和G2-C71碱基对是Pernix TrpRS特异识别的碱基。这些结果表明，ARS对tRNA的识别机制在三种王朝之间是不同的。较少

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