Dynamics of Eukaryotic Translation Initiation

真核翻译起始动力学

• 批准号: 8919425
• 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/8919425
• 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; 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）：该项目旨在定义蛋白质合成 - 翻译 - 在真核生物中启动的动态分子机制。受调节的翻译对于细胞的功能至关重要。蛋白质必须用空间和时间精度合成，以确保细胞活力。相反，翻译不正当 对人类健康的后果，对包括癌症，病毒感染，发育缺陷和自闭症在内的许多疾病至关重要。翻译的启动是其最受调节的相位，是一个复杂的过程，涉及核糖体亚基，mRNA和至少23个多肽，可指导伸长能力的80年代核糖体颗粒的形成。在K99阶段，将在一个指导的环境中开发研究早期翻译启动的现有方法，该设置将扩大O'Leary博士实施单分子技术的能力并开发与真核细胞合作所需的技能。我们将使用单分子荧光显微镜来确定启动过程的关键阶段的动力学 - 生物分子组成和构象的时间进化。拟议的研究基于单分子平台O'Leary Dr. O'Leary开发的，目的是研究启动的最早部分 - 酿酒酵母cerevisiae Cap结合蛋白EIF4E对mRNA 5'CAP的识别，并由预点启动络合物的其他组件对此过程进行调节。我们将扩展这项技术，以发现核糖体扫描的机制，核糖体扫描的机制，mRNA启动密码子所定的过程。我们将开发扫描速率的测定法，并使用它来确定mRNA 5'未翻译区域和启动因子对扫描过程的影响（AIM 1）。我们将定义mRNA-蛋白质相互作用在协调扫描过程中的作用，并更广泛地定义启动动力学（AIM 2）。这些K99相研究将使用酿酒酵母翻译成分作为模型系统进行。尽管酵母是建立起始机制基本原理的宝贵模型，但在将用酵母获得的知识应用于人类翻译时，酵母和人类翻译机制之间存在差异。在R00阶段，我们将通过使用指导阶段中开发的技能以及目标1和2的知识来解决这些差异。为此，在R00阶段，我们将重新建立人类翻译起始机械并表征关键机械差异（AIM 3）。目标1-3的综合结果将提供询问人类健康中心重要的监管机制所需的机械理解（AIM 4）。特别是，我们将检查microRNA和mRNA降解的翻译控制作用。在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，以实现R00阶段及以后的研究人员的独立性。我们的研究结果将为细胞功能的基本方面提供新的见解，并定义与生物学和医学相关的新范式。