Ohtsuka E, Doi T, Fukumoto R, Matsugi J, Ikehara M
Nucleic Acids Res. 1983 Jun 25;11(12):3863-72. doi: 10.1093/nar/11.12.3863.
E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.
使用有限量的核糖核酸酶A水解大肠杆菌甲硫氨酸起始转运核糖核酸（tRNAMetf），得到两个缺少反密码子三聚体和3'-末端二聚体的半分子。通过用多核苷酸激酶加ATP进行磷酸化，然后用RNA连接酶处理，将化学合成的三聚体CUAp和UUAp连接到5'-半分子上。这些修饰的tRNAMetf物种具有与终止密码子UAG和UAA互补的反密码子。通过类似的程序将两个半片段连接起来，得到一个缺少反密码子三聚体和3'-二聚体的分子。在CAU插入的对照tRNAMetf接受甲硫氨酸的条件下，测试了这些tRNA的甲硫氨酸接受活性。发现所有三个修饰分子均未被来自大肠杆菌的甲硫氨酰-tRNA合成酶识别。其他16种氨基酸未与部分纯化的氨酰-tRNA合成酶结合。
