The Cellular and Molecular Effects of Synonymous Mutations

同义突变传统上被认为是沉默的，因为它们不会改变 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， MRNAs的稳定性和/或功能。同义突变与癌症和克罗恩病有关 疾病，抗生素抗药性的发展，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，以及 依赖于计算预测的调查往往无法确定原因 健身效果。此外，非同义突变的影响经常通过新陈代谢和 细胞中的调控网络；没有理由认为同义突变只会影响 只编码了信使核糖核酸，其他什么都没有。之前没有任何研究涉及到系统范围内的影响 同义突变。该项目的目标是1)识别高影响的同义突变 提高大肠杆菌在强选择压力下的适合性，以及2)阐明 这些适合性在系统范围和分子水平上的影响。 目标1描述了将所有可能的同义突变引入300个大肠杆菌编码基因 代谢酶和转录调节因子--总共有312,000个突变。突变的细胞将是 在几种不同的选择压力下进行筛选，以确定高影响的突变。它的巨大规模 Screen让这个项目有别于之前许多专注于轻微有害物质的调查 个体基因的突变。 目的2解决30个高影响同义突变对编码的mRNAs水平的影响 以及蛋白质，目标是识别10个可能通过不同机制发挥作用的蛋白质。全系统范围内 这10个突变的影响将被调查，以帮助我们理解为什么每个突变都能提高适应性。 在特定条件下。 目的3描述10个高影响突变影响的分子机制的研究。 编码mRNAs的结构、稳定性和功能。可能的机制包括创建新的 转录起始点，小调节RNA结合的改变，影响 信使核糖核酸的稳定性和/或翻译，以及编码蛋白结构的变化 翻译的速度，这会影响蛋白质折叠。 这个项目将提供一个前所未有的观察有益的频率和健身效果 同义突变和对10个或更多高冲击影响的详细机制理解 以不同方式影响信使核糖核酸结构、稳定性和功能的同义突变。

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.