Bruce A G, Uhlenbeck O C
Biochemistry. 1982 Mar 2;21(5):855-61. doi: 10.1021/bi00534a007.
An efficient procedure for the replacement of the anticodon and the adjacent hypermodified nucleotide (residues 34-37) of yeast tRNAPhe with any desired oligoribonucleotide sequence has been developed. The four residues are removed by chemical cleavage at Y-37 and partial ribonuclease A digestion at U-33. An oligonucleotide is inserted in three steps by using T4 RNA ligase and T4 polynucleotide kinase. When different oligonucleotides are inserted, both the size of the loop and the sequence of nucleotides in the anticodon region of this tRNA can be varied. The ability of the different anticodon loop substituted tRNAs to be aminoacylated by yeast phenylalanyl-tRNA synthetase is dependent upon the sequence of the oligonucleotide inserted. This suggests that there is an important interaction between the anticodon region of yeast tRNAPhe and its synthetase.
已开发出一种有效的方法，可将酵母苯丙氨酸tRNA的反密码子及相邻的超修饰核苷酸（第34 - 37位残基）替换为任何所需的寡核糖核苷酸序列。通过在Y - 37处进行化学切割以及在U - 33处进行部分核糖核酸酶A消化来去除这四个残基。使用T4 RNA连接酶和T4多核苷酸激酶，通过三个步骤插入寡核苷酸。当插入不同的寡核苷酸时，该tRNA的环大小和反密码子区域的核苷酸序列均可改变。不同反密码子环取代的tRNA被酵母苯丙氨酰 - tRNA合成酶氨酰化的能力取决于插入的寡核苷酸序列。这表明酵母苯丙氨酸tRNA的反密码子区域与其合成酶之间存在重要的相互作用。
