Expanding the Genetic Code with Phosphothreonine and Phosphotyrosine

合成含有遗传密码外氨基酸的蛋白质最近重新成为一个非常感兴趣的话题，并在智力和技术上提出了巨大的挑战。重编程遗传密码需要两个关键事件：(i)创造一种新的高效的“正交”氨基酰tRNA合成酶：tRNA对能够单独产生非规范的氨基酰tRNA。（ii）通过重新编码特定的mRNA密码子来指定该氨基酸插入蛋白质的位置的能力。基于天然存在的两种古细菌氨基酰基-tRNA合成酶样蛋白（O-phosphoseryl -tRNA合成酶及其同源tRNACys和pyrolyyl -tRNA合成酶及其同源tRNAPyl），将基于信使RNA中的无义密码子UGA和UAG，在多肽的预定位置上进行磷苏氨酸和磷酪氨酸的体内共翻译插入，研究开发大肠杆菌系统。鉴于磷苏氨酸和磷酪氨酸在细胞增殖、细胞信号传导和细胞死亡所必需的真核蛋白质中的重要性，一种强大的制造含有磷氨基酸的蛋白质的方法将具有广泛的应用和重要意义。这个项目产生的更广泛的影响是：(i)将要开发的实验工具将在未来被生命科学的其他研究人员广泛使用。开发可行的专利将有助于商业技术。（三）本项目将为本科生和博士后提供跨学科的培训。

Synthesis of proteins containing amino acids outside of the genetic code has recently resurfaced as a topic of much interest and presents a great challenge intellectually and technically. Reprogramming the genetic code requires two key events: (i) The creation of a new efficient 'orthogonal' aminoacyl tRNA synthetase:tRNA pair able to generate solely a non-canonical aminoacyl tRNA. (ii) The ability to specify the position in which this amino acid will be inserted in the protein by recoding a particular mRNA codon. Based on the natural existence of two archaeal aminoacyl-tRNA synthetase-like proteins (O-phosphoseryl tRNA synthetase with its cognate tRNACys and pyrrolysyl-tRNA synthetase with its cognate tRNAPyl) research will be initiated to develop an Escherichia coli system for the in vivo co translational insertion of phosphothreonine and phosphotyrosine at pre-determined positions in a polypeptide based on the nonsense codons UGA and UAG in a messenger RNA. Given the importance of phosphothreonine and phosphotyrosine in eukaryotic proteins essential for cell proliferation, cellular signaling, and cell death, a robust method of making proteins containing phospho-amino acids will have wide application and great significance. The broader impacts resulting from this project are: (i) The experimental tools to be developed will be widely employed in the future by other researchers in the life sciences. (ii) Development of a viable patent will contribute to commercial technology. (iii) This project will provide interdisciplinary training for undergraduate and postdoctoral students.