Ohuchi Masaki, Murakami Hiroshi, Suga Hiroaki
Department of Advanced Iterdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 461 Komaba, Meguroku, Tokyo 1538904, Japan.
Nucleic Acids Symp Ser (Oxf). 2007(51):115-6. doi: 10.1093/nass/nrm058.
Flexizymes are artificial RNA catalysts that enable us to readily prepare aminoacyl-tRNAs with a variety of amino acid and tRNA kinds. On the other hand, because their flexibility feature lacking high specificities toward amino acids and tRNAs, the in situ aminoacylation in a translation apparatus have not been able to executed. We here present a novel strategy to overcome this specificity problem to tRNA using a cis-acting flexizyme-tRNA construct, called a catalytic precursor tRNA, combining with a naturally occurring ribozyme, ribonuclease P (RNase P). In this coupling system of two RNA enzymes, self-aminoacylation occurs on the catalytic precursor tRNA for specific charging of amino acids at the 3'end of the tRNA domain in the presence of the cognate amino acid substrates. Subsequently, the aminoacylated catalytic precursor tRNA is specifically cleaved at the 5'linker region of the tRNA domain, giving the mature aminoacyl-tRNA. Most importantly, the generated flexizyme does not function in trans to tRNAs present in the translation apparatus, indicating that this two-ribozyme coupling system would potentially act as an orthogonal aminoacylation system in the translation apparatus.
柔性酶是一种人工RNA催化剂，能够让我们轻松制备出含有各种氨基酸和tRNA种类的氨酰tRNA。另一方面，由于其灵活性特点，即对氨基酸和tRNA缺乏高特异性，翻译装置中的原位氨酰化作用一直无法实现。我们在此提出一种新策略，通过使用一种顺式作用的柔性酶-tRNA构建体（称为催化前体tRNA），并结合天然存在的核酶核糖核酸酶P（RNase P），来克服tRNA的这种特异性问题。在这两种RNA酶的偶联系统中，在同源氨基酸底物存在的情况下，催化前体tRNA上会发生自我氨酰化，从而在tRNA结构域的3'端对氨基酸进行特异性加载。随后，氨酰化的催化前体tRNA在tRNA结构域的5'连接区被特异性切割，生成成熟的氨酰tRNA。最重要的是，生成的柔性酶不会对翻译装置中存在的tRNA进行反式作用，这表明这种双核酶偶联系统可能会在翻译装置中作为一种正交氨酰化系统发挥作用。
