Hecht S M, Alford B L, Kuroda Y, Kitano S
J Biol Chem. 1978 Jul 10;253(13):4517-20.
Incubation of abbreviated tRNA's (tRNA-C-COH's) with (chemically) preaminoacylated P1, P2-di(adenosine 5'-)diphosphates in the presence of purified RNA ligase effected transfer of an aminoacyladenylate moiety to the 3'-terminus of the abbreviated tRNA's in good yield. Aminoacylated (or misacylated) tRNA's may thus be prepared from fractionated or unfractionated tRNA-C-COH's; each of the five aminoacylated dinucleoside diphosphates tested was utilized as a substrate by RNA ligase. That the resulting "chemically aminoacylated" tRNA's were identical with those prepared by enzymatic aminoacylation was judged by comparison of 1) chromatographic properties on benzolated diethylaminoethyl-cellulose, 2) rates of chemical deacylation, and 3) affinities for elongation factor Tu, as well as 4) the ability of misacylated tRNA's so derived to be deacylated chemically and then reactivated enzymatically with their cognate amino acids.
在纯化的RNA连接酶存在的情况下，将简化的tRNA（tRNA-C-COH）与（化学）预氨酰化的P1、P2-二（腺苷5'-）二磷酸一起温育，可使氨酰腺苷酸部分以良好的产率转移到简化tRNA的3'-末端。因此，可以从分级分离或未分级分离的tRNA-C-COH制备氨酰化（或错氨酰化）的tRNA；所测试的五种氨酰化二核苷二磷酸中的每一种都被RNA连接酶用作底物。通过比较1）在苯甲酰化二乙氨基乙基纤维素上的色谱性质、2）化学脱酰化速率、3）对延伸因子Tu的亲和力以及4）如此衍生的错氨酰化tRNA被化学脱酰化然后用其同源氨基酸进行酶促再活化的能力，判断所得的“化学氨酰化”tRNA与通过酶促氨酰化制备的tRNA相同。
