A general approach for the generation of orthogonal tRNAs
A general approach for the generation of orthogonal tRNAs
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DOI：
10.1016/s1074-5521(01)00063-1
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发表时间：
2001-09-01
影响因子：
--
通讯作者：
Schultz, PG
中科院分区：
文献类型：
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作者：
Wang, L;Schultz, PG
背景资料：向大肠杆菌的遗传密码中添加新的氨基酸需要正交抑制子tRNA，该正交抑制子tRNA通过正交氨酰-tRNA合成酶被所需的非天然氨基酸独特地酰化。从詹氏甲烷球菌（Methanococcus jannaschii）输入的tRNA（CUA）（Tyr）-酪氨酰tRNA合成酶对可用于产生这样的对。体内选择已经被开发用于选择具有增强的正交性的突变型抑制性tRNA，其可用于位点特异性地将非天然氨基酸掺入到E.结果：构建了AY.产生詹氏tRNA（Tyr）。tRNA（CUA）（Tyr）是内源性大肠杆菌的底物。基于芽孢杆菌RNA酶基因中琥珀无义突变的抑制，通过阴性选择从库中删除大肠杆菌氨酰-tRNA合成酶。然后选择剩余的tRNATyr在同源M存在下抑制β-内酰胺酶基因中琥珀无义密码子的能力.詹氏酪氨酰-tRNA合成酶（TyrRS）。选择了四种突变的抑制性tRNA，它们是大肠杆菌合成酶比M. jannaschii tRNAC（CUA）（Tyr），但仍然可以被M.结论：突变型抑制基因tRNA（CUA）（Tyr）与M.詹氏TyrRS是用于将非天然氨基酸体内掺入蛋白质中的优良正交tRNA-合成酶对。这种一般方法可以扩展到产生额外的正交tRNA合成酶对，以及探测tRNA和它们的同源合成酶之间的相互作用。(C)2001爱思唯尔科技有限公司版权所有。
Background: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(CUA)(Tyr)-tyrosyl tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli.Results: A library of amber suppressor tRNAs derived from AY. jannaschii tRNA(Tyr) was generated. tRNA(CUA)(Tyr) s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA Tyr s were then selected for their ability to suppress amber nonsense codons in the beta -lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E coli synthetases than M. jannaschii tRNAC(CUA)(Tyr) but still can be charged efficiently by M. jannaschii TyrRS.Conclusions: The mutant suppressor tRNA(CUA)(Tyr) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases. (C) 2001 Elsevier Science Ltd. All rights reserved.