Studies of Transfer RNA

项目摘要 蛋白质通常由20种氨基酸合成，但结果在蛋白质中发现了300多种氨基酸 翻译后修饰（PTMs）这些天然的非典型氨基酸（ncAA）调节蛋白质的 功能和控制基本的细胞过程。令人满意的ncAA遗传编码需要 通过设计，开发了有效和准确的氨酰-tRNA形成和递送到核糖体的方法， tRNA、tRNA合成酶和构成正交翻译系统（OTS）的延伸因子。而 一些ncAA已经被遗传编码（例如，N-乙酰赖氨酸，磷酸丝氨酸（Sep）），OTS尚未被 为一些关键PTM建立了。本提案的总体目标是通过开发 OTS用于容易和精确地生产天然和工程化蛋白质， 合成ncAA。 这些总目标将在拟议工作的三个具体领域实现。(1)硒的形式 硒代半胱氨酸（Se）是人体必需的微量元素，具有多种有益的化学活性， 其错误掺入的性质涉及许多疾病状态。我们将设计高效的现场指导 插入Sec，并研究其插入沿着其前体Sep进入几种酶的影响。 工业和医疗利益。(2)虽然遗传密码曾被认为是通用的， 自然界中的自然现象现在已经普遍存在。我们将把生物信息学分析与我们的 tRNA身份元件的知识，以揭示新的遗传密码，并更好地表征这种作用， 自然界的变异性。此外，我们将使用长期进化来产生具有新遗传密码的生物体 利用合成氨基酸。(3)我们计划创建氨酰-tRNA合成酶，用于高效合成ncAA- tRNA为一系列磷酸氨基酸和化学反应性合成氨基酸。考虑到 磷酸化在细胞信号传导和激酶抑制剂对癌细胞的成功，并根据我们的 成功地建立了磷酸丝氨酸的OTS，我们建议建立额外的磷酸氨基的OTS 酸及其不可水解的类似物。化学反应性氨基酸的掺入将提供稳健的 将PTM、生物物理探针或其他有价值的残基引入目标蛋白质的工具。 拟议的工作是重要的，因为生产，纯化，生物化学和结构的能力， 在确定位点含有ncAA的acrylate蛋白对于阐明基本细胞过程是必不可少的 以及构建蛋白质设计的新工具。这项工作的创新之处在于， 这些生物学相关的ncAA，并为生物化学和生物医学研究人员提供有效的OTS， 揭示PTM的复杂网络及其在生物技术和人类健康中的作用。

PROJECT SUMMARY 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) modulate 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 ncAA- tRNA 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 char- acterize 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.