大肠杆菌琥珀抑制tRNA基因的构建。III. tRNA特异性的测定。

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity.

作者信息

Normanly J, Kleina L G, Masson J M, Abelson J, Miller J H

机构信息

Department of Biology, California Institute of Technology, Pasadena 91125.

出版信息

J Mol Biol. 1990 Jun 20;213(4):719-26. doi: 10.1016/S0022-2836(05)80258-X.

DOI:10.1016/S0022-2836(05)80258-X

PMID:2141650

Abstract

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.

摘要

我们使用合成寡核苷酸构建了一组大肠杆菌琥珀抑制tRNA基因。为了确定它们的特异性，这些tRNA分别用于抑制大肠杆菌二氢叶酸还原酶基因fol中的琥珀（UAG）无义突变。对突变蛋白进行纯化并进行N端序列分析，以确定抑制tRNA在琥珀密码子位置插入了哪种氨基酸。根据蛋白质序列信息，这些抑制子可分为三组。I类抑制子，tRNA（CUAAla2）、tRNA（CUAGly1）、tRNA（CUAHisA）、tRNA（CUALys）和tRNA（CUAProH），插入了预测的氨基酸。II类抑制子，tRNA（CUAGluA）、tRNA（CUAGly2）和tRNA（CUAIle1），被谷氨酰胺氨酰tRNA合成酶部分或主要错误充电。III类抑制子，tRNA（CUAArg）、tRNA（CUAAspM）、tRNA（CUAIle2）、tRNA（CUAThr2）、tRNA（CUAMet（m））和tRNA（CUAVal）主要插入赖氨酸。