We recently described an orthogonal initiator tRNA (itRNA^Ty2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNA^Ty2 (itRNA^Ty2_AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNA^Ty2_AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNA^Ty2_AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.

中文翻译：

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使用重新编程的启动子和无义密码子对三种不同的非规范氨基酸进行遗传编码

我们最近描述了一种正交启动子tRNA（i tRNA ^Ty2），它可以响应UAG无义密码子，用非规范氨基酸（ncAAs）启动蛋白质合成。在这里，我们报告的一个突变的i tRNA ^Ty2（i tRNA ^Ty2 _AUA）可以有效地启动翻译响应UAU酪氨酸密码子，从而在其N端产生带有ncAA的蛋白质。我们表明，在表达i tRNA ^Ty2 _AUA的细胞中，UAU可以用作双重用途密码子，在起始位置选择性编码ncAA，在延长位置选择性编码酪氨酸。使用i tRNA ^Ty2 _AUA，结合其同源的酪氨酰-tRNA合成酶和两个相互正交的吡咯基-tRNA合成酶，我们证明UAU可以与UAG或UAA一起重新分配，以在同一蛋白质中编码两个不同的ncAA。此外，通过工程化一个吡咯基-tRNA合成酶之一的底物特异性，我们开发了一个三重正交系统，该系统能够同时重新分配UAU，UAG和UAA，以在精确定义的位点产生包含三种不同ncAA的蛋白质。为了展示该系统的实用性，我们生产了包含两个或三个ncAA且具有独特的生物正交官能团的蛋白质，并证明了这些蛋白质可以用多个荧光探针分别修饰。