Dynamics of Eukaryotic Translation Initiation

描述(申请人提供)：该项目旨在确定在真核生物中启动蛋白质合成-翻译-的动态分子机制。受调控的翻译是细胞功能的基础；蛋白质必须在空间和时间上精确合成，以确保细胞的活力。相比之下，监管不善的翻译情况却很糟糕 对人类健康的影响，是许多疾病的核心，包括癌症、病毒感染、发育缺陷和自闭症。翻译的启动是其最受调控的阶段，是一个复杂的过程，涉及核糖体亚单位、mRNA和至少23个多肽，这些多肽指导着一个具有伸长能力的80S核糖体颗粒的形成。在K99阶段，研究早期翻译启动的现有方法将在有指导的环境中发展，这将扩大奥利里博士实施单分子技术的能力，并发展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动力学--生物分子组成和构象的时间演化--这是启动过程的关键阶段的基础。这项拟议的研究建立在奥利里博士开发的单分子平台上，目的是研究酿酒酵母帽子结合蛋白eIF4E对mRNA5‘端帽子的最早部分的起始识别，以及引发前复合体的其他成分对这一过程的调节。我们将扩展这项技术，以揭示核糖体扫描的机制，即mRNA起始密码子的定位过程。我们将开发一种扫描速度的分析方法，并用它来确定mRNA5‘非翻译区和启动因子对扫描过程的影响(目标1)。我们将具体定义mRNA-蛋白质相互作用在协调扫描过程中的作用，以及更一般的启动动力学(目标2)。这些K99阶段的研究将使用酿酒酵母翻译成分作为模型系统进行。虽然酵母是建立启动机制基本原理的宝贵模型，但在将酵母获得的知识应用于人工翻译时，必须考虑酵母和人工翻译机制之间的差异。在R00阶段，我们将通过使用在指导阶段发展的技能以及目标1和2产生的知识来解决这些差异。为此，在R00阶段，我们将重组人工翻译启动机制，并描述关键机制差异(目标3)。目标1-3的综合结果将提供审问对人类健康至关重要的重要调控机制所需的机械性理解(目标4)。特别是，我们将研究通过microRNAs和mRNA降解进行翻译控制的作用。在K99阶段获得的指导支持、技能和数据的结合将为奥利里博士提供一个跳板，使他在R00阶段及以后成为一名独立的研究人员。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。

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.