Roesser J R, Xu C, Payne R C, Surratt C K, Hecht S M
Department of Chemistry, University of Virginia, Charlottesville 22901.
Biochemistry. 1989 Jun 13;28(12):5185-95. doi: 10.1021/bi00438a041.
Several pyroglutamylaminoacyl-tRNA's were prepared by T4 RNA ligase mediated condensation of synthetic pyroglutamylaminoacyl-pCpA's with tRNA's from which the last two nucleotides at the 3'-end had been removed. The derived pyroglutamylaminoacyl-tRNA's were incubated in the presence of calf liver pyroglutamate aminopeptidase, which effected their conversion to free aminoacyl-tRNA's. The lack of contaminating esterase activities in the pyroglutamate aminopeptidase was verified by direct assay for the presence of the aminoacyl moieties in the formed aminoacyl-tRNA's and by the use of the deblocked aminoacyl-tRNA's as acceptors in the peptidyltransferase reaction using an Escherichia coli ribosomal system. These findings provide the wherewithal for a detailed investigation of the substrate specificity of the peptidyltransferase center and for the elaboration of polypeptides containing modified amino acids at predetermined sites.
通过T4 RNA连接酶介导的合成焦谷氨酰氨基酰 - pCpA与3'-末端最后两个核苷酸已被去除的tRNA的缩合反应，制备了几种焦谷氨酰氨基酰 - tRNA。将得到的焦谷氨酰氨基酰 - tRNA在小牛肝焦谷氨酸氨基肽酶存在下孵育，该酶使其转化为游离氨基酰 - tRNA。通过直接测定形成的氨基酰 - tRNA中氨基酰部分的存在，以及使用去封闭的氨基酰 - tRNA作为受体在大肠杆菌核糖体系统的肽基转移酶反应中，验证了焦谷氨酸氨基肽酶中不存在污染性酯酶活性。这些发现为详细研究肽基转移酶中心的底物特异性以及在预定位点合成含修饰氨基酸的多肽提供了条件。
