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

古细菌氨酰tRNA合成酶识别tRNA机制的研究

• 批准号: 13680705
• 负责人: HASEGAWA Tsunemi
• 金额: $ 2.24万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680705/
• 关键词: Archaea identity; Archaea; Aeropyrum pernix K1; Aminoacyl-tRNA synthetase; Evolution; Threonine tRNA; Proline tRNA; Tryptophan tRNA; Aeropyrum pernix; トリプトファニル-tRNA合成酵素; トリプトファンRNA; 分子進化; チロシル-tRNA合成酵素; チロシンtRNA; in vitro転写反応; プロリル-tRNA合成酵素

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

尽管在包括大肠杆菌（大肠杆菌）在包括大肠杆菌（大肠杆菌）的大细菌系统中广泛研究了tRNA同一性元素，但对古细菌的氨基酰基-TRNA合成酶（ARS）的tRNA识别位点知之甚少，古细菌被归类为第三王国。为了研究苏氨酸，脯氨酸和色氨酸tRNA的分子机制，用于极端嗜热古老的AROPyrum Pernix K1的每个ARS，每个ARS基因都被克隆并过表达。研究了使用体外野生和突变型TRNA转录物以及过表达ARS的氨基酰化实验。在苏氨酸系统中，结果支持了我们先前的发现，即a.pernix thrrs无法识别苏氨酸tRNA的歧视剂基础。该结果表明歧视基础在古细菌中的重要性多样性，因为来自卤素考古的苏氨酸tRNA的歧视基础，haloferax volcanii，是同源性的强大决定因素。还发现，尽管有N末端截短的独特结构，但A. Pernix Thrrs确实识别了AC…苏氨酸tRNA和其他物种的更多Ceptor茎区域。此外，显示出脯氨酸tRNA的体外转录本，其第二和第三对的受体茎被突变为C-G，除了通过a.pernix thrrs屈服于G36U的移植。通过仅移植五个碱基，从脯氨酸tRNA转换为苏氨酸tRNA的结果表明，苏氨酸threonine tRNA的主要身份元素是受体茎区域和反密码子。在脯氨酸系统中，发现鉴别碱基A73，G1-C72碱基对以及反密码单子G35和G36是a.pernix prors的强确定剂。 G1-C72碱基对的识别与E.Coli Prors系统完全不同。在色氨酸系统中，结果表明，反密码子C34，C35和A36，鉴别剂g73，G1-C62和G2-C71base对受体茎端的基端是由A.Pernix TRPR专门识别的。这些结果表明，在三个王国中，ARS的tRNA识别机制不同。较少的

项目成果

Nagaoka, Y., Yokozawa, J., Umehara, T., Iwaki, J., Okamoto, K., Kawarabayasi, Y., Koyama, Y., Sako, Y., Wakagi, T., Kuno, A., Hasegawa, T.: "Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyr

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Kikuchi, K., Umehara, T., Fukuda, K., Hwang, J., Kuno, A., Hasegawa, T.: "RNA aptamers targeted domain II of hepatitis C IRES could bind its apical loop region"Journal of Biochemistry. 133・3. 263-270 (2003)

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