Molecular Evolution of RNA modification enzymes and RNA recognition mechanism

在这个项目中，我们在RNA修饰领域获得了很多发现。主要研究结果如下。(1)高温下体外RNA修饰体系的构建我们已经明确了嗜热细菌特异性多胺对RNA修饰机制的作用。(2) RNA修饰酶RNA识别机制的阐明我们报道了Aquifex aeolicus TrmD [tRNA （m1 G37）甲基转移酶]具有独特的RNA识别机制。该酶能甲基化具有G36G37序列和A36G37序列的tRNA分子。此外，该酶甲基化具有A36G37序列的微螺旋RNA。这些结果表明，与其他TrmD蛋白相比，风衣曲霉TrmD具有广泛的底物特异性。我们报道真核生物tRNA （m7G46）甲基转移酶[Trm8/Trm82复合物]识别tRNA中的d臂结构，表明真核生物酶与细菌酶相比具有严格的RNA识别机制。(3)阐明蛋白质结构与RNA识别机制的关系我们对TrmH [tRNA （Gm18）甲基转移酶]中保守的碱性氨基酸残基进行了定点诱变研究。我们可以区分第一个tRNA结合过程中需要的残基和催化循环过程中需要的残基。

In this project, we have obtained many findings in the RNA modification field.Main findings are as follows.(1) Construction of in vitro RNA modification system under high temperaturesWe have clarified roles of thermophilic-bacteria specific polyamines against the RNA modification machinery.(2) Clarifications of RNA recognition mechanisms of RNA modification enzymesWe have reported that Aquifex aeolicus TrmD [tRNA (m1 G37) methyltransferase] has a unique RNA recognition mechanism.The enzyme methylates tRNA molecules possessing not only G36G37 sequence but also A36G37 sequence. Furthermore, the enzyme methylates micro-helix RNA possessing A36G37 sequence. These results showed that A. aeolicus TrmD has a broad substarte specificity in comparison with other TrmD proteins.We have reported that eukaryote tRNA (m7G46) methyltransferase [Trm8/Trm82 complex] recognizes the D-arm structure in tRNA, demonstrating that eukaryote enzyme has a strict RNA recognition mechanism as compared to bacterial enzymes.(3) Clarifications of relationship between protein structure and RNA recognition mechanismWe performed site-directed mutagenesis study against basic amino acid residues conserved in TrmH [tRNA (Gm18) methyltransferases]. We could distinguish residues required in the first tRNA binding process and residues required in the progress of the catalytic cycles.