Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs:: concomitant suppression of three different termination codons in an mRNA in mammalian cells
Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs:: concomitant suppression of three different termination codons in an mRNA in mammalian cells
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DOI：
10.1093/nar/gkh959
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
RajBhandary, UL
中科院分区：
文献类型：
--
作者：
Köhrer, C;Sullivan, EL;RajBhandary, UL
我们描述了一套完整的正交第 21 合成酶-琥珀、赭石和蛋白石抑制 tRNA 对的生成，包括第 21 合成酶-赭石抑制 tRNA 对的首次报告。我们发现，源自大肠杆菌谷氨酰胺 tRNA 的琥珀、赭色和蛋白石抑制 tRNA 分别抑制哺乳动物细胞报告基因 mRNA 中的 UAG、UAA 和 UGA 终止密码子。每种抑制性 tRNA 的活性取决于大肠杆菌谷氨酰胺酰-tRNA 合成酶的表达，表明哺乳动物细胞质中二十种氨酰-tRNA 合成酶均不会氨酰化任何抑制性 tRNA。通过在抑制 tRNA 基因中引入进一步的突变，产生了具有广泛抑制活性（分别增加高达 36 倍、156 倍和 200 倍）的琥珀、赭石和蛋白石抑制 tRNA。最活跃的抑制 tRNA 已被组合使用，以同时抑制 mRNA 中的两个或三个终止密码子。我们讨论了这些第 21 种合成酶抑制 tRNA 对的潜在用途，用于将两个或什至三个不同的非天然氨基酸定点掺入蛋白质中，以及用于哺乳动物细胞中琥珀、赭石和蛋白石终止密码子的调节抑制。
We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.