The Cellular and Molecular Effects of Synonymous Mutations

同义突变的细胞和分子效应

• 批准号: 9926908
• 负责人: SHELLEY D. COPLEY
• 金额: $ 31.2万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926908
• 关键词: Address; Affect; Amino Acids; Antibiotic Resistance; Bacteria; Bar Codes; Binding; Biochemical Pathway; CRISPR/Cas technology; Carbon; Cells; Codon Nucleotides; Computer Models; Crohn' s disease; DNA Sequence Alteration; Development; Engineering; Environment; Enzymes; Escherichia coli; Evolution; Frequencies; Genes; Genetic Transcription; Genetic Translation; Genome; Glucose; Glutamates; Goals; Growth; High-Throughput Nucleotide Sequencing; Individual; Investigation; Label; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Messenger RNA; Metabolic; Methods; Molecular; Mutation; Nitrogen; Phenotype; Plasmids; Play; Poison; Population; Proline; Proteins; Proteome; Quantitative Reverse Transcriptase PCR; RNA; Ribosomal Proteins; Role; Salmonella enterica; Salmonella typhimurium; Source; Structure; System; Transcription Initiation Site; Translations; Untranslated RNA; cell type; fitness; high throughput screening; human disease; in vivo; mRNA Stability; metabolome; mutant; nonsynonymous mutation; novel; pleiotropism; pressure; promoter; protein folding; protein structure; recombinase; transcriptome

Synonymous mutations have traditionally been considered to be silent because they do not change the encoded amino acid. However, evidence is mounting that synonymous mutations can alter the structure, stability and/or function of mRNAs. Synonymous mutations have been implicated in cancer and Crohn’s disease, the development of antibiotic resistance, and bacterial adaptation to novel conditions. Few studies have investigated the mechanistic basis of the effects of synonymous mutations on mRNA structure, and investigations that have relied on computational predictions have often failed to identify the reasons for fitness effects. Further, the effects of non-synonymous mutations often ripple through the metabolic and regulatory networks in cells; there is no reason to think that synonymous mutations will affect only the encoded mRNA and nothing else. No previous study has addressed the system-wide effects of synonymous mutations. The goals of this project are 1) to identify high-impact synonymous mutations that increase the fitness of E. coli under strong selective pressures, and 2) to elucidate the mechanistic basis of these fitness effects at the system-wide and molecular levels. Aim 1 describes the introduction of all possible synonymous mutations into 300 E. coli genes encoding metabolic enzymes and transcriptional regulators – a total of 312,000 mutations. The mutant cells will be screened under several different selective pressures to identify high-impact mutations. The vast scale of this screen sets this project apart from many previous investigations that have focused on mildly deleterious mutations in individual genes. Aim 2 addresses the effects of 30 high-impact synonymous mutations on the levels of the encoded mRNAs and proteins with the goal of identifying 10 that likely operate via different mechanisms. The system-wide effects of these 10 mutations will be investigated to help us understand why each mutation increases fitness under specific conditions. Aim 3 describes investigations of the molecular mechanisms by which 10 high-impact mutations affect the structure, stability and function of the encoded mRNAs. Possible mechanisms include creation of new transcriptional start sites, alteration of the binding of small regulatory RNAs, changes in structure that affect mRNA stability and/or translation, and changes in the structure of an encoded protein due to alterations of the tempo of translation, which can affect protein folding. This project will provide an unprecedented look at the frequencies and fitness effects of beneficial synonymous mutations and a detailed mechanistic understanding of the effects of 10 or more high-impact synonymous mutations that affect mRNA structure, stability and function in different ways.

传统上，同义突变被认为是沉默的，因为它们不会改变 编码的氨基酸。但是，有证据表明合成突变可以改变结构， mRNA的稳定性和/或功能。癌症和克罗恩的同义突变已隐含 疾病，抗生素耐药性的发展以及对新疾病的根本适应。很少的研究 已经研究了同义突变对mRNA结构的影响的机理基础， 放弃计算预测的调查通常未能确定 健身效果。此外，非同义突变的影响通常通过代谢和 细胞中的调节网络；没有理由认为同义突变只会影响 编码mRNA，别无其他。以前没有研究解决了全系统范围的影响 同义突变。该项目的目标是1）确定高影响力的同义突变 增加大肠杆菌在强选择性压力下的适应性，2）阐明机械基础 这些适应性在整个系统和分子水平上的影响。 AIM 1描述了将所有可能的同义突变引入300大肠杆菌基因编码的 代谢酶和转录调节剂 - 总计312,000个突变。突变细胞将是 在几个不同的选择压力下进行筛选，以识别高影响力突变。大规模的 屏幕集将该项目设置为许多以前的调查，这些调查重点是删除 单个基因的突变。 AIM 2解决了30个高影响力合成突变对编码mRNA水平的影响 蛋白质的目标是识别10种可能通过不同机制运行的蛋白质。整个系统 将研究这10个突变的影响，以帮助我们了解为什么每个突变都会增加健身 在特定条件下。 AIM 3描述了对10个高影响突变影响分子机制的研究 编码的mRNA的结构，稳定性和功能。可能的机制包括创建新的 转录启动位点，小调节RNA的结合的改变，影响的结构变化 mRNA稳定性和/或翻译，以及由于改变而改变的编码蛋白结构的变化 翻译的速度，会影响蛋白质折叠。 该项目将对有益的频率和健身效应提供前所未有的观察 同义突变和对10个或更多高影响力影响的详细机械理解 以不同方式影响mRNA结构，稳定性和功能的同义突变。

项目成果

The physical basis and practical consequences of biological promiscuity.

• DOI: 10.1088/1478-3975/ab8697
• 发表时间: 2020-04-03
• 期刊: Physical biology
• 影响因子: 2

Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.

• DOI: 10.1073/pnas.2316834121
• 发表时间: 2024-01
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Dong-Dong Yang-Dong;Leo M. Rusch;Karl A Widney;A. Morgenthaler;S. Copley