T4 RNA连接酶介导的新型“化学错酰化”苯丙氨酸tRNA的制备。
T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS.
作者信息
Heckler T G, Chang L H, Zama Y, Naka T, Chorghade M S, Hecht S M
出版信息
Biochemistry. 1984 Mar 27;23(7):1468-73. doi: 10.1021/bi00302a020.
T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer"). The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose. Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E. coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation .
T4 RNA连接酶用于使缺失胞苷-75和腺苷-76的大肠杆菌苯丙氨酸tRNA（tRNAPhe-COH；受体“寡聚物”）与几种化学酰化的对氯苯甲酰腺苷（pCpA）衍生物（供体“寡聚物”）中的每一种进行缩合。在DEAE-纤维素和苯甲酰化DEAE-纤维素上进行色谱处理后，得到的“化学错配酰化”苯丙氨酸tRNA（tRNAPheS）的产率为20%-65%。通过以下方法对化学错配酰化的tRNA进行表征：（i）在将“错误”氨基酸进行化学脱酰化后，用大肠杆菌苯丙氨酰-tRNA合成酶使化学错配酰化的苯丙氨酸tRNA与苯丙氨酸进行酶促再氨基酰化；（ii）比较Cu2+溶液对化学和酶促制备的N-乙酰-L-苯丙氨酰-tRNAPheS样品的水解作用；（iii）测量化学错配酰化产生的tRNA种类的色谱行为。
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