Dynamics of Eukaryotic Translation Initiation

真核翻译起始动力学

• 批准号: 8765942
• 负责人: Sean E O'Leary
• 金额: $ 9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765942
• 关键词: 5' Untranslated Regions; 7-methylguanosine; Accounting; Address; Architecture; Autistic Disorder; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Biology; Cell Cycle Progression; Cell physiology; Cells; Complex; Data; Defect; Development; Disease; Ensure; Eukaryota; Eukaryotic Cell; Event; Evolution; Fluorescence Microscopy; Goals; Health; Human; Initiator Codon; Kinetics; Knowledge; Label; Malignant Neoplasms; Measurement; Mediating; Medicine; Mentors; Messenger RNA; Methodology; Methods; MicroRNAs; Modeling; Molecular; Molecular Conformation; Paint; Pathway interactions; Peptide Initiation Factors; Phase; Positioning Attribute; Process; Property; Protein Biosynthesis; Proteins; RNA Cap-Binding Proteins; RNA Recognition Motif; Regulation; Research; Research Personnel; Ribosomes; Role; Saccharomyces cerevisiae; Scanning; Signal Transduction; Staging; Stimulus; Structure; System; Techniques; Technology; Time; Translating; Translation Initiation; Translations; Untranslated Regions; Virus Diseases; Work; Yeasts; biophysical techniques; experience; helicase; human disease; insight; knowledge of results; mRNA Transcript Degradation; molecular dynamics; particle; polypeptide; public health relevance; reconstitution; scaffold; single molecule; skills

DESCRIPTION (provided by applicant): This project aims to define the dynamic molecular mechanism by which protein synthesis - translation - is initiated in eukaryotes. Regulated translation is fundamental to the function of the cell; proteins must be synthesized with spatial and temporal precision to ensure cellular viability. In contrast, misregulated translation has dire consequences for human health and is central to many diseases including cancer, viral infection, developmental defects, and autism. Initiation of translation is its most regulated phase and is a complex process involving the ribosomal subunits, mRNA, and at least 23 polypeptides that guide the formation of an elongation-competent 80S ribosomal particle. In the K99 phase, existing methodologies to study early translation initiation will be developed in a mentored setting that expands Dr. O'Leary's abilities to implement single-molecule techniques and develops skills needed to work with eukaryotic cells. We will use single- molecule fluorescence microscopy to determine the dynamics - the time-evolution of biomolecular composition and conformation - that underpin key phases of the initiation process. The proposed research builds on the single-molecule platform Dr. O'Leary has developed to study the earliest part of initiation - recognition of the mRNA 5' cap by the Saccharomyces cerevisiae cap-binding protein eIF4E and the modulation of this process by other components of the pre-initiation complex. We will expand this technology to uncover the mechanism of ribosomal scanning, the process through which the mRNA start codon is located. We will develop an assay for the rate of scanning and use this to determine the effects of the mRNA 5' untranslated region and initiation factors on the scanning process (Aim 1). We will define the role of mRNA-protein interactions in coordinating the scanning process specifically, and the dynamics of initiation more generally (Aim 2). These K99-phase studies will be carried out using S. cerevisiae translation components as a model system. While yeast is an invaluable model for establishing the fundamentals of the initiation mechanism, there are differences between the yeast and human translation machinery that must be taken into account when applying knowledge obtained with yeast to human translation. In the R00 phase, we will address these differences by using the skills developed during the mentored phase and the knowledge resulting from Aims 1 and 2. To this end, in the R00 phase we will reconstitute the human translation initiation machinery and characterize key mechanistic differences (Aim 3). The combined results from Aims 1 - 3 will provide the mechanistic understanding needed to interrogate important regulatory mechanisms central to human health (Aim 4). In particular, we will examine the roles of translational control by microRNAs and mRNA degradation. The combination of mentored support, skills, and data obtained in the K99 phase will provide Dr. O'Leary a springboard to achieving independence as a researcher in the R00 phase and beyond. The results of our studies will provide new insights into fundamental aspects of cellular function, and will define new paradigms relevant to biology and medicine.

描述（由申请人提供）：该项目旨在定义真核生物中启动蛋白质合成（翻译）的动态分子机制。调节翻译是细胞功能的基础。蛋白质的合成必须具有空间和时间精度，以确保细胞活力。相比之下，翻译监管不当会带来可怕的后果。 它对人类健康产生影响，并且是许多疾病的核心，包括癌症、病毒感染、发育缺陷和自闭症。翻译起始是其最受调控的阶段，是一个复杂的过程，涉及核糖体亚基、mRNA 和至少 23 个多肽，指导形成具有延伸能力的 80S 核糖体颗粒。在 K99 阶段，研究早期翻译起始的现有方法将在指导环境下开发，以扩展 O'Leary 博士实施单分子技术的能力，并培养使用真核细胞所需的技能。我们将使用单分子荧光显微镜来确定支撑启动过程关键阶段的动力学——生物分子组成和构象的时间演化。拟议的研究建立在 O'Leary 博士开发的单分子平台上，用于研究起始的最早部分 - 酿酒酵母帽结合蛋白 eIF4E 对 mRNA 5'帽的识别以及其他成分对该过程的调节预引发复合物。我们将扩展这项技术来揭示核糖体扫描的机制，即定位 mRNA 起始密码子的过程。我们将开发一种扫描速率测定方法，并用它来确定 mRNA 5' 非翻译区和起始因子对扫描过程的影响（目标 1）。我们将明确 mRNA-蛋白质相互作用在协调扫描过程中的作用，以及更广泛的启动动态（目标 2）。这些 K99 阶段研究将使用酿酒酵母翻译组件作为模型系统进行。虽然酵母是建立启动机制基础的宝贵模型，但在将酵母获得的知识应用于人类翻译时，必须考虑酵母和人类翻译机器之间的差异。在 R00 阶段，我们将通过使用指导阶段开发的技能以及目标 1 和 2 产生的知识来解决这些差异。为此，在 R00 阶段，我们将重建人工翻译启动机制并表征关键的机制差异（目标 3）。目标 1 - 3 的综合结果将提供探究对人类健康至关重要的重要监管机制（目标 4）所需的机制理解。特别是，我们将研究 microRNA 和 mRNA 降解对翻译控制的作用。 K99 阶段获得的指导支持、技能和数据的结合将为 O'Leary 博士提供一个跳板，使其成为 R00 阶段及以后的研究人员实现独立。我们的研究结果将为细胞功能的基本方面提供新的见解，并将定义与生物学和医学相关的新范式。