Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
J Am Chem Soc. 2010 Feb 24;132(7):2142-4. doi: 10.1021/ja9068722.
The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by aminoacyl-tRNA synthetase/tRNA pairs, which direct the unique coupling of specific amino acids with specific anticodons. The evolutionary record suggests that a primitive genetic code expanded into the current genetic code, over billions of years, through duplication and specialization (neofunctionalization) of aminoacyl-tRNA synthetases and tRNAs from common ancestral synthetase/tRNA pairs. This process produced the current set of mutually orthogonal aminoacyl-tRNA synthetases and tRNAs that direct natural protein synthesis. Here we demonstrate the creation of new orthogonal pairs, which are mutually orthogonal with existing orthogonal pairs, de novo, by a logical series of steps implemented in the laboratory, via the de novo generation of orthogonality in RNA-RNA interactions, protein-RNA interactions, and small molecule substrate selection by protein catalysts. Our laboratory evolution experiments provide experimental evidence for duplication and specialization as a plausible route to the current set of synthetases and tRNAs via natural evolution. Moreover our experiments extend billions of years of natural evolution and demonstrate that the small number of naturally occurring orthogonal aminoacyl-tRNA synthetase/tRNA pairs do not place an intrinsic limitation on the scope of synthetic genetic code expansion for the incorporation of multiple distinct unnatural amino acids into proteins or the synthesis and evolution of unnatural polymers in cells.
遗传密码规定了密码子与蛋白质翻译中编码的氨基酸之间的对应关系。该密码由氨酰-tRNA 合成酶/tRNA 对强制执行，它们指导特定氨基酸与特定反密码子的独特结合。进化记录表明，一个原始的遗传密码通过氨酰-tRNA 合成酶和 tRNA 的复制和特化（新功能化），在数十亿年的时间里扩展成了现在的遗传密码。这个过程产生了目前相互正交的氨酰-tRNA 合成酶和 tRNA，它们指导自然蛋白质合成。在这里，我们通过实验室中一系列逻辑步骤，通过 RNA-RNA 相互作用、蛋白质-RNA 相互作用和蛋白质催化剂对小分子底物的选择中的新的正交性的从头产生，展示了新的正交对的创建，这些正交对与现有的正交对是相互正交的。我们的实验室进化实验为复制和特化提供了实验证据，证明了它们是通过自然进化达到目前的合成酶和 tRNA 集合的一种合理途径。此外，我们的实验扩展了数十亿年的自然进化，并表明天然存在的少数正交氨酰-tRNA 合成酶/tRNA 对不会对将多个不同的非天然氨基酸掺入蛋白质或在细胞中合成和进化非天然聚合物的合成遗传密码扩展的范围造成内在限制。
