Studies of Transfer RNA

转移RNA的研究

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要（家长奖 R35GM122560）蛋白质通常由 20 种氨基酸合成，但蛋白质中含有 300 多种氨基酸，例如翻译后修饰 (PTM) 的结果。这些天然非规范氨基酸 (ncAA) 调制分析蛋白质功能并控制基本细胞过程。令人满意的 ncAA 遗传编码需要开发高效且准确的氨酰基-tRNA 形成和递送至核糖体的方法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 已被基因编码（例如 N-乙酰赖氨酸、磷酸丝氨酸） (Sep))，尚未为许多关键 PTM 建立 OTS。该提案的总体目标是通过开发 OTS 来重新连接翻译，以便轻松、精确地生产天然和工程物质含有天然和合成 ncAA 的蛋白质。这些总体目标将分三期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对人类健康有益的元素，通过错误掺入表现出许多有利的化学特性与许多疾病状态有关。我们将设计高效的 Sec 定点插入并进行调查其与其前体 Sep 一起插入几种具有工业和医学意义的酶中。 (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现在已知已广泛传播。我们将生物信息分析与我们对 tRNA 身份的了解结合起来元素既能揭示新的遗传密码，又能更好地表征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抗癌细胞的成功，基于我们成功建立磷酸丝氨酸 OTS，我们建议建立以下 OTS：额外的磷酸氨基酸及其不可水解的类似物。掺入化学反应性物质氨基酸将提供强大的工具来引入 PTM、生物物理探针或其他有价值的残基转化为感兴趣的蛋白质。拟议的工作意义重大，因为能够生产、纯化、对特定位点含有 ncAA 的蛋白质进行生化和结构表征对于阐明基本细胞过程并构建蛋白质设计新工具。这拟议工作的创新之处在于对这些生物学相关的 ncAA 进行基因编码，并提供为生化和生物医学研究人员提供高效的 OTS，帮助解开 PTM 的复杂网络及其在生物技术和人类健康中的作用。

项目成果

期刊论文数量（56）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Diversification of aminoacyl-tRNA synthetase activities via genomic duplication.

通过基因组复制实现氨酰-tRNA 合成酶活性的多样化。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Krahn, Natalie;Söll, Dieter;Vargas
通讯作者：
Vargas

Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids.

用于体内编码 D-氨基酸的遗传密码扩展工具包的合理设计。

DOI：
发表时间：
2023
期刊：
影响因子：
0
作者：
Jiang, Han;Weng, Jui;Wang, Yi;Tsou, Jo;Chen, Pei;Ko, An;Söll, Dieter;Tsai, Ming;Wang, Yane
通讯作者：
Wang, Yane

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNAHis pair.

基因组修饰的大肠杆菌菌株，携带正交大肠杆菌组氨酰-tRNA 合成酶 – tRNAHis 对。

DOI：
发表时间：
2017-11
期刊：
影响因子：
0
作者：
Englert, Markus;Vargas;Reynolds, Noah M;Wang, Yane;Söll, Dieter;Umehara, Takuya
通讯作者：
Umehara, Takuya

Transfer RNAs with novel cloverleaf structures.

具有新颖的三叶草结构的转移RNA。

DOI：
发表时间：
2017
期刊：
影响因子：
14.9
作者：
Mukai, Takahito;Vargas;Englert, Markus;Tripp, H James;Ivanova, Natalia N;Rubin, Edward M;Kyrpides, Nikos C;Söll, Dieter
通讯作者：
Söll, Dieter

Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli.

大肠杆菌将位点特异性硒代半胱氨酸掺入蛋白质的挑战。