Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria
Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria
复制标题
DOI：
10.1073/pnas.031488298
复制
发表时间：
2001-02-27
影响因子：
11.1
通讯作者：
RajBhandary, UL
中科院分区：
文献类型：
--
作者：
Kowal, AK;Köhrer, C;RajBhandary, UL
开发用于在体内将氨基酸类似物位点特异性掺入蛋白质的方法的两个关键要求是（i）不被任何内源性氨酰-tRNA合成酶（aaRS）氨酰化的抑制性tRNA和（ii）氨酰化抑制性tRNA但不氨酰化细胞中的其它tRNA的氨酰-tRNA合成酶。在这里，我们描述了两个这样的aaRS抑制tRNA对，一个用于酵母酿酒酵母和另一个用于大肠杆菌。“第21对合成酶-tRNA对”包括E.大肠杆菌酪氨酰-tRNA合成酶（GlnRS）沿着来自人起始tRNA的琥珀抑制子，用于酵母，和酵母酪氨酰-tRNA合成酶（TyrRS）的突变体沿着来自大肠杆菌的琥珀抑制子。coli启动子tRNA，用于E.杆菌抑制性tRNA仅在异源aaRS存在下在体内被氨酰化，并且氨酰化的tRNA有效地抑制琥珀密码子。质粒携带E. coli GlnRS基因在酵母中能稳定表达。然而，携带酵母TyrRS基因的质粒不能在E.杆菌这种稳定性的缺乏很可能是由于野生型酵母TyrRS错氨基酰化E. coli脯氨酸tRNA。利用易错PCR技术，我们分离并鉴定了三个能在大肠杆菌中稳定表达的酵母TyrRS突变体。杆菌这些突变体在体内仍然基本上定量地氨酰化抑制性tRNA，但在体外对抑制性tRNA的区分力比E.大肠杆菌脯氨酸tRNA的因子为2.2- 6.8倍。
Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can he stably expressed in E. coli. These mutants still aminoacylates the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.