Identification and Conversion of Molecular Recognition Elements in Yeast Tyrosine tRNA

(1)将酵母酪氨酸tRNA应用于大肠杆菌的无细胞蛋白质合成系统中。大肠杆菌中，它被证明是带有赖氨酸的轻微电荷。大肠杆菌赖氨酰-tRNA合成酶（LysRS）。因此，本项目的主要目标是寻找一种方法来保护tRNA不被E. coli LysRS. (2)酵母酪氨酸tRNA的几种变体具有潜在的抗E.用酵母酪氨酰-tRNA合成酶（TyrRS）和大肠杆菌（E. coli）测定了这些tRNA变异体的氨酰化活性。coliLysRS与赖氨酸的错酰化。(3)一种变体tRNA，其中反密码子茎中的3个A-U碱基对被3个G-C碱基对取代，被证明在错误酰化中急剧减少，同时保持原始的酪氨酸接受能力。从而证明这些变异实际上是反对E的负恒等式的一部分。coli LysRS. (4)利用基因工程技术改造酵母TyrRS的氨基酸特异性，并取得了实质性进展。(5)通过在酵母酪氨酸tRNA的反密码子中插入一个碱基（2-氨基-6-（2-噻吩基）嘌呤）和在转录的ras mRNA中插入另一个碱基（吡啶-2-酮），开发了非天然（非沃森-克里克型）碱基对。

(1) When yeast tyrosine tRNA was used in a cell-free protein-synthesizing system derived from E. coli, it was shown to be charged slightly with lysine by E. coli lysyl-tRNA synthetase (LysRS). Therefore, the primary target of this project was set to find out a way to protect the tRNA from mis-acylation by E. coli LysRS.(2) Several variants of yeast tyrosine tRNA, which have potentially negative identity elements against E. coli LysRS, were synthesized by our new method using transcription with T7 RNA polymerase and processing with RNase P. Aminoacylation activities of these tRNA variants were measured with yeast tyrosyl-tRNA synthetase (TyrRS) for normal tyrosine acceptance and E. coli LysRS for mis-acylation with lysine.(3) A variant tRNA, in which 3 A-U base pairs in the anticodon stem were replaced with 3 G-C base pairs, was shown to be drastically reduced in the mis-acylation, while keeping the original tyrosine-accepting ability. Thus these variations are proved to be actually part of the negative identity elements against E. coli LysRS.(4) Conversion of the amino acid specificity of yeast TyrRS was attempted by using genetic engineering and a substantial progress was achieved.(5) An unnatural (non-Watson-Crick type) base pair was developed by inserting one base (2-amino-6-(2-thienyl)purine) in the anticodon of yeast tyrosine tRNA and the other one (pyridin-2-one) in the transcribed ras mRNA.