Rodriguez Erik A, Lester Henry A, Dougherty Dennis A
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
RNA. 2007 Oct;13(10):1715-22. doi: 10.1261/rna.667607. Epub 2007 Aug 13.
The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased DeltaG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS', which shows approximately 50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.
将非天然氨基酸掺入蛋白质中是添加生物物理探针、生物正交功能基团和光反应性交联剂的一种有价值的工具，尽管这些方法通常需要难以通过化学氨酰化tRNA获得的蛋白质量。THG73是一种琥珀色抑制tRNA，已被广泛使用，在20种蛋白质中掺入了100多个残基。体外研究表明，大肠杆菌天冬酰胺琥珀色抑制tRNA（ENAS）的抑制效果优于THG73。然而，我们在此报告，在非洲爪蟾卵母细胞中，ENAS的抑制效率不到THG73的26%。然后，我们测试了新开发的嗜热四膜虫谷氨酰胺琥珀色抑制tRNA（TQAS）文库，该文库在受体茎的第二至第四位含有突变。受体茎突变对抑制效率没有不利影响，实际上还可以提高抑制效率。组合突变导致抑制效率平均化，并且抑制效率的提高与受体茎ΔG的增加无关。我们创建了一种嗜热四膜虫乳白抑制tRNA，TQOpS'，其相对于THG73显示出约50%的抑制效率。由功能性抑制tRNA组成的TQAS tRNA文库已经创建，这将允许在真核细胞中进行筛选，而在真核细胞中对大型文库进行快速分析是不可行的。
