Englisch-Peters S, Conley J, Plumbridge J, Leptak C, Söll D, Rogers M J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Biochimie. 1991 Dec;73(12):1501-8. doi: 10.1016/0300-9084(91)90184-3.
This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.
本文聚焦于大肠杆菌谷氨酰胺-tRNA合成酶（GlnRS）和tRNA（Gln）突变体特异性的几个方面。先前已描述了位于GlnRS基因glnS中的温度敏感突变体。分析了导致温度敏感表型的突变，并对这些突变体的假回复突变体进行了分离和表征。根据这些突变在tRNA（Gln）-GlnRS复合物三维结构中的位置，讨论了这些突变的性质。为了表征氨酰化反应的特异性，合成了以2'-脱氧AMP或3'-脱氧AMP作为其3'-末端核苷酸的突变体tRNA（Gln）种类。随后的氨酰化和ATP/PPi交换活性测定确定了谷氨酰胺与末端腺苷的2'-羟基酯化；3'-OH基团没有谷氨酰胺化。这与GlnRS作为I类氨酰-tRNA合成酶的分类相关。讨论了tRNA（Gln）中影响GlnRS识别的突变，并综述了大肠杆菌中谷氨酰胺同一性的当前概念。
