Studies of Transfer RNA

转移RNA的研究

• 批准号: 10553492
• 负责人: DIETER SOLL
• 金额: $ 39.01万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553492
• 关键词: Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Apoptosis; Area; Award; Biochemical; Bioinformatics; 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; Parents; Pharmacology; Phosphoamino Acids; Phosphorylation; Phosphoserine; Post-Translational Protein Processing; Process; Production; Protein Engineering; Proteins; Research Personnel; Ribosomes; Role; Route; Selenium; Selenocysteine; Series; Signal Transduction; Site; 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 (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.

项目总结(父奖项R35GM122560) 蛋白质通常由20个氨基酸合成，但在蛋白质中发现超过300个氨基酸，如 翻译后修饰(PTM)的结果。这些天然的非规范氨基酸(NCAA)是- Uate蛋白质的功能和控制基本的细胞过程。NCAA令人满意的遗传编码 需要开发高效和准确的氨基酰-tRNA的形成和输送到核糖体 通过设计tRNA、tRNA合成酶和构成正交翻译的延伸因子 系统(OTS)。虽然一些NCAA已经被遗传编码(例如，N-乙酰赖氨酸、磷丝氨酸 (9月))，尚未为一些关键的PTM建立OTS。这项提案的总体目标是 通过开发OTS重新连接翻译，以方便和精确地生产自然和工程设计的产品 含有天然的和人工合成的NCAA的蛋白质。这些总体目标将在三年内实现 拟议工作的具体领域。(1)硒以硒半胱氨酸(SEC)的形式存在，是一种必需的痕量元素 有益于人类健康的元素，由于它的错误结合而显示出许多有利的化学性质 牵涉到许多疾病状态。我们将设计高效的现场定向插入SEC并调查 它与其前体Sep一起插入到几种具有工业和医学价值的酶中的效果。 (2)虽然遗传密码曾经被认为是通用的，但自然界中的自然密码子重新分配是普遍存在的 现在已经广为人知了。我们将把生物信息学分析与我们的tRNA鉴定知识结合起来 这些元素既揭示了新的遗传密码，又更好地表征了这种变异性在自然界中的作用。 此外，我们将使用长期进化来产生具有新遗传密码的有机体 合成氨基酸。(3)我们计划创造氨基酰-tRNA合成酶来有效地合成 NcAAtRNA用于一系列磷酸氨基酸和化学反应的合成氨基酸。给定 磷酸化在细胞信号转导中的关键作用和激酶抑制剂抗癌细胞的成功， 在我们成功建立了磷化丝氨酸OTS的基础上，我们建议建立OTS 附加的磷酸氨基酸及其非水解性类似物。掺入化学反应性 氨基酸将提供一个强大的工具来引入PTM、生物物理探针或其他有价值的残基 变成一种感兴趣的蛋白质。拟议的工作意义重大，因为生产、提纯、 生化和结构特征的蛋白质在特定的位置含有NCAA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。这个 拟议工作的创新是对这些具有生物相关性的NCAA进行基因编码，并提供 生化和生物医学研究人员帮助解开PTMS复杂网络的高效OTS 以及它们在生物技术和人类健康方面的作用。

项目成果

Ancestral archaea expanded the genetic code with pyrrolysine.

祖先古细菌用吡咯赖氨酸扩展了遗传密码

• DOI: 10.1016/j.jbc.2022.102521
• 发表时间: 2022-11
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Guo, Li -Tao;Amikura, Kazuaki;Jiang, Han -Kai;Mukai, Takahito;Fu, Xian;Wang, Yane-Shih;O'Donoghue, Patrick;Soell, Dieter;Tharp, Jeffery M.
• 通讯作者: Tharp, Jeffery M.

Engineering aminoacyl-tRNA synthetases for use in synthetic biology.

• DOI: 10.1016/bs.enz.2020.06.004
• 发表时间: 2020
• 期刊: The Enzymes
• 作者: Krahn N;Tharp JM;Crnković A;Söll D
• 通讯作者: Söll D

Measuring the tolerance of the genetic code to altered codon size.

• DOI: 10.7554/elife.76941
• 发表时间: 2022-03-16
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: DeBenedictis, Erika Alden;Soll, Dieter;Esvelt, Kevin M.
• 通讯作者: Esvelt, Kevin M.

Initiating protein synthesis with noncanonical monomers in vitro and in vivo.

• DOI: 10.1016/bs.mie.2021.05.002
• 发表时间: 2021
• 期刊: Methods in enzymology
• 作者: Tharp JM;Walker JA;Söll D;Schepartz A
• 通讯作者: Schepartz A

Mistranslation of the genetic code by a new family of bacterial transfer RNAs.

• DOI: 10.1016/j.jbc.2023.104852
• 发表时间: 2023-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Schuntermann, Dominik B.;Fischer, Jonathan T.;Bile, Jonmatthew;Gaier, Sarah A.;Shelley, Brett A.;Awawdeh, Aya;Jahn, Martina;Hoffman, Kyle S.;Westhof, Eric;Soell, Dieter;Clarke, Christopher R.;Vargas-Rodriguez, Oscar
• 通讯作者: Vargas-Rodriguez, Oscar