Initiating protein synthesis with noncanonical monomers in vitro and in vivo.
Initiating protein synthesis with noncanonical monomers in vitro and in vivo.
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DOI：
10.1016/bs.mie.2021.05.002
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发表时间：
2021
影响因子：
--
通讯作者：
Schepartz A
中科院分区：
文献类型：
--
作者：
Tharp JM;Walker JA;Söll D;Schepartz A
除了少数例外，核糖体蛋白质合成开始于蛋氨酸（或其衍生物N-甲酰基-蛋氨酸），跨越所有生命领域。甲硫氨酸作为起始氨基酸的作用是由其同源tRNA的独特结构决定的，称为tRNAfMet。通过错误酰化tRNAfMet，我们和其他人已经表明，蛋白质合成可以在体外和体内用各种典型和非典型氨基酸启动。此外，由于起始氨基酸的α-胺不是形成肽键所必需的，因此可以用与传统α-氨基酸几乎没有相似性的各种结构上不同的羧酸来起始翻译。在此，我们提供了一个详细的协议，开始在体外蛋白质合成取代苯甲酸和1，3-二羰基化合物。这些部分通过错酰化的tRNAfMet引入肽的N-末端，错酰化的tRNAfMet通过flexizyme催化的tRNA酰化制备。此外，我们描述了一个协议，开始在体内蛋白质合成与芳香族非典型氨基酸（ncAAs）。该方法依赖于工程化的嵌合起始物tRNA，其通过正交氨酰-tRNA合成酶用ncAA酰化。总之，这些系统是有用的平台，用于生产N-末端修饰的蛋白质和工程化的蛋白质合成机制的大肠杆菌接受额外的非蛋白质羧酸单体。
With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNAfMet. By mis-acylating tRNAfMet, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNAfMet, prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers.
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影响因子：
18.2
作者：
Ad, Omer;Hoffman, Kyle S.;Schepartz, Alanna
通讯作者：
Schepartz, Alanna
影响因子：
4.1
作者：
Bhattacharyya, Souvik;Varshney, Umesh
通讯作者：
Varshney, Umesh
影响因子：
4.5
作者：
Goto, Yuki;Murakami, Hiroshi;Suga, Hiroaki
通讯作者：
Suga, Hiroaki
DOI：
10.1073/pnas.1711482114
发表时间：
2017-11-07
影响因子：
11.1
作者：
McMurry, Jonathan L.;Chang, Michelle C. Y.
通讯作者：
Chang, Michelle C. Y.
影响因子：
8.4
作者：
Dumas A;Lercher L;Spicer CD;Davis BG
通讯作者：
Davis BG