Studies of Transfer RNA

转移RNA的研究

基本信息

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
项目类别：
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

项目摘要

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.

项目概要蛋白质通常由 20 种氨基酸合成，但蛋白质中含有超过 300 种氨基酸翻译后修饰（PTM）。这些天然非规范氨基酸 (ncAA) 调节蛋白质发挥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高效、准确的氨酰基-tRNA 形成并递送至核糖体 tRNA、tRNA 合成酶和延伸因子构成正交翻译系统 (OTS)。尽管一些 ncAA 已被基因编码（例如，N-乙酰赖氨酸、磷酸丝氨酸 (Sep)），OTS 尚未被编码为许多关键 PTM 建立。该提案的总体目标是通过开发重新连接翻译 OTS 用于轻松、精确地生产天然和工程蛋白质，其中含有天然存在的和合成 ncAA。这些总体目标将在拟议工作的三个具体领域中实现。 (1) 硒，形式硒代半胱氨酸（Sec），是人体健康必需的微量元素，具有许多有利的化学性质其错误掺入与许多疾病状态有关。我们将设计高效的现场定向插入 Sec 并研究其插入及其前体 Sep 对几种酶的影响工业和医疗兴趣。 (2) 虽然遗传密码曾经被认为是通用的，但自然密码子现在已知自然界中的重新分配是普遍存在的。我们将生物信息分析与我们的 tRNA 识别元件的知识不仅可以揭示新的遗传密码，还可以更好地表征其作用性质的可变性。此外，我们将利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) 我们计划创建氨酰基-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。鉴于关键作用细胞信号传导中的磷酸化以及激酶抑制剂对抗癌细胞的成功，并基于我们的成功建立磷酸丝氨酸的 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加入将提供强大的将 PTM、生物物理探针或其他有价值的残基引入感兴趣的蛋白质的工具。拟议的工作意义重大，因为生产、纯化、生化和结构特征的能力表征在特定位点含有 ncAA 的蛋白质对于阐明基本细胞过程至关重要以及构建蛋白质设计的新工具。该工作的创新之处在于基因编码这些生物学相关的 ncAA，并为生化和生物医学研究人员提供有效的 OTS，以帮助揭示 PTM 的复杂网络及其在生物技术和人类健康中的作用。