TRANSLATION SYSTEM WITH COMPLEXITY

我们证明了密码子CUG在几个物种中同时被分配为丝氨酸和亮氨酸密码子，使用tRNA分析和遗传技术。为了评估在Candid菌株中多义解码的生物学意义，我们正在解决负责tRNA的亮氨酸接受的定量操作。我们已经证明，亮氨酸的接受是由反密码子37位附近的1-甲基鸟苷决定的。此外，73位的A也参与了亮氨酰化反应。本课题中，我们从大肠杆菌中制备了m1 G的突变和修饰酶的各种转录物。coli中表达。此外，丝氨酰-tRNA和亮氨酰-tRNA合成酶也在E. coli中进行纯化。通过测定使用突变体的亮氨酰化反应的酶促参数，似乎37位的甲基对于亮氨酸 * 是必不可少的。验收此外，在位置73处的突变G至A使Kcat/Km值增加400倍。基于这些观察，我们将这些突变基因导入念珠菌细胞，并评估细胞功能的改变。

We demonstrated that codon CUG is assigned simultaneously both-as serine arid leucine codons in several- Candida-species, using analyzes of tRNA-and genetic techniques. In order to evaluate biological significance of the polysemous decoding in Candid strains, we are addressing quantitative manipulation of leucine acceptance of responsible tRNA. We already showed that leucine acceptance is governed by the presence of 1-methylguanosine at the position 37 adjacent to the anticodon. Moreover it is suggested that A at 73 position is also significantly involved in leucylation. In this project/we prepared various transcripts with mutation and modification enzyme for m1G from E. coli by overexpression method. Furthermore, seryl-tRNA and leucyl-tRNA synthetases were also expressed in E. coli and purified. By determination of enzymatic parameters of leucylation reaction using mutants, it appeared that methyl group at position 37 is indispensable for leucine *. Acceptance. Furthermore, mutation G to A at position 73 increased Kcat/Km values 400-fold. Base upon these observations, we will introduce these mutants genes into Candida cells and alternation of cell function will be evaluated.