Studies of Transfer RNA

项目概要（母合同R35 GM 122560） 蛋白质通常由20种氨基酸合成，但在蛋白质中发现超过300种氨基酸， 翻译后修饰（PTMs）。这些天然非典型氨基酸（ncAAs）mod- 改变蛋白质功能并控制基本的细胞过程。ncAA的满意遗传编码 需要开发有效和准确的氨酰-tRNA形成和递送到核糖体 通过设计构成正交翻译的tRNA、tRNA合成酶和延伸因子 系统（OTS）虽然一些ncAA已经被遗传编码（例如，N-乙酰赖氨酸，磷酸丝氨酸 （9月）），但尚未为一些关键的PTM建立OTS。本提案的总体目标是 通过开发OTS来改造翻译，以方便和精确地生产天然和工程 含有天然存在和合成ncAA的蛋白质。这些总体目标将在三个方面实现 拟议工作的具体领域。(1)硒以硒代半胱氨酸（Sec）的形式存在，是人体必需的微量元素 人体健康的元素，表现出许多有利的化学性质与其错误的掺入 与许多疾病有关。我们将设计有效的定点插入Sec，并研究 影响其与其前体Sep一起沿着插入到几种工业和医学上感兴趣的酶中。 (2)虽然遗传密码曾被认为是普遍的，但自然界中的天然密码子重置是普遍的。 现在已经广泛传播。我们将结合生物信息学分析和我们对tRNA身份的了解， 这些元素既揭示了新的遗传密码，又更好地描述了这种变异在自然界中的作用。 此外，我们将利用长期进化来产生一种具有新遗传密码的生物体， 合成氨基酸(3)我们计划创建氨酰-tRNA合成酶，用于高效合成 ncAAtRNA为一系列磷酸氨基酸和化学反应性合成氨基酸。鉴于 磷酸化在细胞信号传导中的关键作用以及激酶抑制剂对癌细胞的成功， 基于我们成功地建立了磷酸丝氨酸的OTS，我们建议建立以下OTS： 另外的磷酸氨基酸及其不可水解的类似物。加入化学反应性 氨基酸将为引入PTM、生物物理探针或其他有价值的残基提供一个强有力的工具 转化成感兴趣的蛋白质所提出的工作是重要的，因为生产，纯化， 生物化学和结构表征在确定位点含有ncAA的蛋白质是必不可少的， 阐明基本的细胞过程和构建蛋白质设计的新工具。的 所提出的工作的创新是对这些生物学相关的ncAA进行遗传编码，并提供 为生物化学和生物医学研究人员提供有效的OTS，以帮助解开复杂的PTM网络 及其在生物技术和人类健康中的作用。

PROJECT SUMMARY (of the parent award R35GM122560) Proteins are typically synthesized with 20 amino acids, yet over 300 amino acids are found in proteins as a result of posttranslational modifications (PTMs). These natural noncanonical amino acids (ncAAs) mod- ulate protein function and control fundamental cellular processes. Satisfactory genetic encoding of ncAAs requires the development of efficient and accurate aminoacyl-tRNA formation and delivery to the ribosome by design of tRNAs, tRNA synthetases, and elongation factors that constitute orthogonal translation systems (OTSs). While some ncAAs have been genetically encoded (e.g., N-acetyllysine, phosphoserine (Sep)), OTSs have not been established for a number of critical PTMs. The overall goal of this proposal is to rewire translation by developing OTSs for facile and precise production of natural and engineered proteins containing naturally occurring and synthetic ncAAs. These general goals will be realized in three specific areas of the proposed work. (1) Selenium, in the form of selenocysteine (Sec), is an essential trace element for human health, exhibiting many advantageous chemical properties with its misincorporation implicated in many disease states. We will engineer efficient site-directed insertion of Sec and investigate the effects its insertion along with its precursor Sep into several enzymes of industrial and medical interest. (2) While the genetic code was once thought to be universal, natural codon reassignments in nature are now known to be widespread. We will couple bioinformatic analysis with our knowledge of tRNA identity elements to both reveal novel genetic codes and better characterize the role of this variability in nature. Additionally, we will use long-term evolution to produce an organism with a new genetic code utilizing synthetic amino acids. (3) We plan to create aminoacyl-tRNA synthetases for efficient synthesis of ncAAtRNA for a series of phosphoamino acids and chemically reactive synthetic amino acids. Given the critical role of phosphorylation in cell signaling and the success of kinase inhibitors against cancer cells, and based on our success establishing an OTS for phosphoserine, we propose to establish OTSs for additional phosphoamino acids and their non-hydrolyzable analogs. Incorporation of chemically reactive amino acids will provide a robust tool to introduce PTMs, biophysical probes, or other valuable residues into a protein of interest. The proposed work is significant because the ability to produce, purify, biochemically and structurally characterize proteins containing ncAAs at defined sites is essential for elucidation of fundamental cellular processes and for construction of new tools for protein design. The innovation of the proposed work is to genetically encode these biologically relevant ncAAs, and provide efficient OTSs for biochemical and biomedical researchers to help unravel the complex network of PTMs and their role in biotechnology and human health.