Studies of Transfer RNA

转移RNA的研究

• 批准号: 9895829
• 负责人: DIETER SOLL
• 金额: $ 103.4万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895829
• 关键词: Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Apoptosis; Area; Biochemical; Bioinformatics; Biological; Biology; Biophysics; Biotechnology; Cell physiology; Cells; Chemicals; Code; Codon Nucleotides; Complex; DNA; Development; Disease; Elements; Elongation Factor; Engineering; Enzymes; Evolution; Exhibits; Gene Expression; Genetic; Genetic Code; Genetic Diseases; Goals; Health; Human; Industrialization; Knowledge; Link; Malignant Neoplasms; Medical; Metabolic Diseases; Molecular; Nature; Neurodegenerative Disorders; Organism; Pharmacology; Phosphoamino Acids; Phosphorylation; Phosphoserine; Post-Translational Protein Processing; Process; Production; Protein Engineering; Proteins; RNA, Transfer, Amino Acid-Specific; Research Personnel; Ribosomes; Role; Route; Salvelinus; Selenium; Selenocysteine; Series; Signal Transduction; Site; Structure; System; Trace Elements; Transfer RNA; Translations; Work; analog; base; cancer cell; cancer genetics; chemical property; design; frontier; human disease; innovation; interest; kinase inhibitor; novel; novel therapeutic intervention; protein function; success; targeted treatment; tool

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.

项目摘要 通常将蛋白质与20个氨基酸合成，但在蛋白质中发现超过300个氨基酸 翻译后修饰（PTMS）。这些天然的非规范氨基酸（NCAA）调节蛋白质 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，开发有效，准确的氨基酰基-TRNA并通过设计 构成正交翻译系统（OTSS）的TRNA，TRNA合成酶和伸长因子。尽管 某些NCAA已经过遗传编码（例如，N-乙酰透析酶，磷酸磷酶（SEP）），OTS尚未是 为许多关键的PTM建立。该提议的总体目标是通过开发来重新翻译翻译 用于自然和工程蛋白质的便利和精确生产的OTS，这些蛋白质包含天然发生的OTS和 合成NCAA。 这些一般目标将在拟议工作的三个特定领域实现。 （1）硒，形式 硒代半胱氨酸（SEC）是人类健康的重要痕迹，表现出许多有利的化学物质 其不当结合的特性与许多疾病状态有关。我们将设计有效的站点定向 插入SEC并研究其插入及其前体SEP的影响 工业和医疗利益。 （2）虽然遗传密码曾经被认为是通用的，但天然密码子 现在已知自然界的重新分配是广泛的。我们将将生物信息学分析与我们的 了解tRNA身份元素以揭示新的遗传代码并更好地表征这一点的作用 自然界的可变性。此外，我们将使用长期进化来生产具有新遗传代码的生物 利用合成氨基酸。 （3）我们计划创建氨基酰基-TRNA合成酶，以有效合成NCAA- 一系列磷酸氨基酸和化学反应性合成氨基酸的tRNA。鉴于关键作用 细胞信号传导中的磷酸化和激酶抑制剂对癌细胞的成功，并基于我们 成功建立磷酸盐OTS，我们提议为其他磷酸氨基建立OTS 酸及其不可用的类似物。化学反应性氨基酸的掺入将提供强大的 将PTM，生物物理探针或其他有价值残基引入感兴趣蛋白质的工具。 提出的工作很重要，因为生产，纯化，生化和结构上的能力 在定义位置含有NCAA的蛋白质对于阐明基本细胞过程至关重要 并用于构建用于蛋白质设计的新工具。拟议工作的创新是遗传编码 这些与生物学相关的NCAA，并为生化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。