Dynamic pathways of eukaryotic translation initiation

真核翻译起始的动态途径

• 批准号: 9327001
• 负责人: JOSEPH D PUGLISI
• 金额: $ 46.19万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327001
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Biochemical; Biology; Bypass; Complex; Data; Development; Disease; Eukaryota; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Future; Gene Expression; Genes; Initiator Codon; Initiator tRNA; Internal Ribosome Entry Site; Knowledge; Label; Length; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Modeling; Molecular Conformation; Monitor; Mutation; Neurodegenerative Disorders; Organism; Pathway interactions; Peptide Initiation Factors; Process; Proteins; RNA; RNA Conformation; Reagent; Recruitment Activity; Regulation; Ribosomal RNA; Ribosomes; Role; Saccharomyces cerevisiae; Scanning; Series; Signal Transduction; Stimulus; Structure; System; Therapeutic Intervention; Time; Transfer RNA; Translation Initiation; Translational Regulation; Translations; Yeasts; cricket paralysis virus; human disease; molecular rearrangement; response; single molecule; single-molecule FRET; time use

PROJECT SUMMARY Initiation of translation in eukaryotic organisms is a complex and regulated process. Canonical initiation involves a coordinated assembly of ribosomal 40S subunit bound with initiator tRNA and trimeric factor eIF2 on the 5'-end of messenger RNAs (mRNAs) containing a 5' cap structure. This process is guided by >10 initiation factors that mediate loading of 40S subunits, subsequent ATP-dependent scanning of the ribosomal complex through a 5' untranslated region until the first start codon is recognized, and finally 60S subunit joining to begin translation. Here, we will delineate the dynamic pathways of translation initiation using multicolor single- molecule fluorescence to track initiation directly in real time. Our approach will use the genetically, biochemically and structurally well-characterized yeast translation system to reveal the real-time composition and conformation of translation initiation complexes. In Aim 1, we will investigate the composition of initiation complexes on 4 model mRNAs that span distinct initiation mechanisms. In Aim 2, we will observe initiation on the same mRNAs using conformational signals monitored by FRET: conformation of the 40S subunit, mRNA, and factors, and their interrelation will be determined using single-molecule methods. In Aim 3, we will delineate the mechanistic pathways of ribosomal scanning through 5'-untranslated regions (UTRs) that differ in length, sequence and structure. We will determine the timing of scanning, and compare results for predicted timescales from different models of scanning; we will also monitor scanning directly, and determine the time-dependent composition of a scanning ribosomal complex. Our central hypothesis is that distinct dynamic branch points exist during initiation, leading to different potential pathways and timescales of initiation. The expected results should provide a dynamic overview of the complex process of initiation, aiding in our understanding of translation in basic biology and disease. RELEVANCE Translation is the endpoint of gene expression and highly regulated. Translational control allows rapid and spatially localized response to stimuli. Disruption of translational regulation has been implicated in many human diseases, such as cancers and neurodegenerative disease. Understanding of initiation mechanisms is essential for future therapeutic intervention.

项目总结 真核生物中翻译的启动是一个复杂且受调控的过程。典则 启动涉及与启动子tRNA结合的核糖体40S亚单位的协调组装 和含有5‘帽的信使RNA(MRNAs)5’端的三聚体因子eIF2 结构。这一过程由介导40s亚基加载的启动因子引导， 随后通过5‘非翻译区对核糖体复合体进行依赖于ATP的扫描 直到第一个起始密码子被识别，最后60S亚基加入开始翻译。 在这里，我们将描绘出多色单色翻译启动的动态路径。 分子荧光，直接实时跟踪引发。我们的方法将使用 遗传、生化和结构特征良好的酵母翻译系统揭示 翻译起始复合体的实时组成和构象。在目标1中，我们将 研究4种跨度不同的模型mRNAs上起始复合体的组成 启动机制。在目标2中，我们将观察在相同的mRNAs上使用 FRET监测的构象信号：40s亚单位、信使核糖核酸和 因素，以及它们之间的相互关系将使用单分子方法确定。在目标3中，我们 将描述核糖体扫描通过5‘非翻译区的机制途径 (UTRs)在长度、序列和结构上不同。我们将确定扫描的时间， 并比较来自不同扫描模型的预测时间尺度的结果；我们还将 直接监视扫描，并确定扫描的随时间变化的组成 核糖体复合体。我们的中心假设是不同的动态分支点存在于 启蒙，导致不同的潜在启蒙路径和时间尺度。预期中的 结果应该提供复杂的启动过程的动态概述，有助于我们的 理解基础生物学和疾病方面的翻译。 相关性 翻译是基因表达的终点，受到高度调控。翻译控制允许 对刺激的快速和空间局部化反应。对翻译监管的破坏一直是 与许多人类疾病有关，如癌症和神经退行性疾病。 了解启动机制对于未来的治疗干预是至关重要的。