Martin F, Reinbolt J, Dirheimer G, Gangloff J, Eriani G
Unité Propre de Recherche 9002 du Centre National de la Recherche Scientifique, Strasbourg, France.
RNA. 1996 Sep;2(9):919-27.
Elements that confer identity to a tRNA in the cellular environment, where all aminoacyl-tRNA synthetases are competing for substrates, may be delineated by in vivo experiments using suppressor tRNAs. Here we describe the selection of active Escherichia coli tRNAAsp amber mutants and analyze their identity. Starting from a library containing randomly mutated tRNA(CUA)Asp genes, we isolated four amber suppressors presenting either lysine, alanine, or glutamine activity. Two of them, presenting mainly alanine or lysine activity, were further submitted to a second round of mutagenesis selection in order to improve their efficiency of suppression. Eleven suppressors were isolated, each containing two or three mutations. Ten presented identities of the two parental mutants, whereas one had switched from lysine to arginine identity. Analysis of the different mutants revealed (or confirmed for some nucleotides) their role as positive and/or negative determinants in AlaRS, LysRS, and ArgRS recognition. More generally, it appears that tRNAAsp presents identity characteristics closely related to those of tRNALys, as well as a structural basis for acquiring alanine or arginine identity upon moderate mutational changes; these consist of addition or suppression of the corresponding positive or negative determinants, as well as tertiary interactions. Failure to isolate aspartic acid-inserting suppressors is probably due to elimination of the important G34 identity element and its replacement by an antideterminant when changing the anticodon of the tRNAAsp to the CUA triplet.
在细胞环境中,所有氨酰 - tRNA合成酶都在竞争底物,赋予tRNA身份的元件可以通过使用抑制tRNA的体内实验来确定。在此,我们描述了活性大肠杆菌tRNAAsp琥珀突变体的筛选,并分析了它们的身份。从包含随机突变的tRNA(CUA)Asp基因的文库开始,我们分离出了四个具有赖氨酸、丙氨酸或谷氨酰胺活性的琥珀抑制子。其中两个主要呈现丙氨酸或赖氨酸活性的抑制子,为了提高它们的抑制效率,进一步进行了第二轮诱变筛选。分离出了11个抑制子,每个抑制子包含两个或三个突变。其中10个呈现出两个亲本突变体的身份特征,而有一个从赖氨酸身份转变为精氨酸身份。对不同突变体的分析揭示了(或对某些核苷酸进行了确认)它们在丙氨酰 - tRNA合成酶(AlaRS)、赖氨酰 - tRNA合成酶(LysRS)和精氨酰 - tRNA合成酶(ArgRS)识别中作为正向和/或负向决定因素的作用。更普遍地说,似乎tRNAAsp呈现出与tRNALys密切相关的身份特征,以及在适度突变变化后获得丙氨酸或精氨酸身份的结构基础;这些包括添加或去除相应的正向或负向决定因素,以及三级相互作用。未能分离出插入天冬氨酸的抑制子可能是由于在将tRNAAsp的反密码子改变为CUA三联体时,重要的G34身份元件被消除并被一个反决定因素所取代。