The Cellular and Molecular Effects of Synonymous Mutations

同义突变的细胞和分子效应

• 批准号: 9367552
• 负责人: SHELLEY D. COPLEY
• 金额: $ 34.53万
• 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 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9367552
• 关键词: Address; Affect; Amino Acids; Antibiotic Resistance; Bacteria; Binding; Biochemical Pathway; CRISPR/Cas technology; Carbon; Cells; Codon Nucleotides; Computer Simulation; 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; killings; mRNA Stability; metabolome; mutant; 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.

同义突变传统上被认为是沉默的，因为它们不改变基因的结构。 编码的氨基酸。然而，越来越多的证据表明同义突变可以改变结构， mRNA的稳定性和/或功能。同义突变与癌症和克罗恩病有关 疾病，抗生素耐药性的发展，以及细菌对新条件的适应。很少有研究 研究了同义突变对mRNA结构影响的机制基础， 依赖于计算预测的调查往往未能确定 健身效果此外，非同义突变的影响往往涟漪代谢和 没有理由认为同义突变只会影响细胞中的基因调控网络， 编码的mRNA，除此之外什么都没有。以前的研究没有涉及到全系统的影响， 同义突变该项目的目标是：1）识别高影响同义突变， 提高E.大肠杆菌在强大的选择压力，和2）阐明的机制基础， 这些健身效果在系统范围和分子水平。 目的1描述了将所有可能的同义突变引入300 E中。大肠杆菌基因编码 代谢酶和转录调节因子-总共312，000个突变。变异细胞将 在几种不同的选择压力下筛选，以确定高影响力的突变。这场大规模的 屏幕设置这个项目除了许多以前的调查，集中在轻度有害 个别基因的突变。 目的2解决了30个高影响同义突变对编码mRNA水平的影响 和蛋白质，目的是确定10种可能通过不同机制起作用的蛋白质。全系统 这10个突变的影响将被调查，以帮助我们了解为什么每个突变增加健身 在特定条件下。 目的3描述了10个高影响力突变影响基因表达的分子机制的研究。 结构，稳定性和功能的编码的mRNA。可能的机制包括建立新的 转录起始位点，小调控RNA结合的改变，影响 mRNA的稳定性和/或翻译，以及由于mRNA的改变而引起的编码蛋白质结构的变化。 翻译的克里思，这会影响蛋白质的折叠。 这个项目将提供一个前所未有的频率和健身效果的有益 同义突变和10个或更多高影响的影响的详细机制的理解 同义突变以不同方式影响mRNA的结构、稳定性和功能。