Dynamics of Eukaryotic Ribosomal Scanning

真核核糖体扫描动力学

基本信息

批准号：
10456244
负责人：
Sean E O'Leary
金额：
$ 31.77万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10456244
关键词：
5&apos Untranslated Regions Address Area Base Sequence Behavior Binding Biochemical Biological Assay Biophysics Cells Chemicals Complement Complex Data Disease Ensure Eukaryota Event Failure Fluorescence Genetic Health Heart Initiator Codon Length Life Location Malignant Neoplasms Measures Messenger RNA Methods Modeling Molecular Motion Nucleotides Open Reading Frames Outcome Peptide Initiation Factors Phase Play Positioning Attribute Process Property Protein Biosynthesis Proteins Proteome RNA Helicase Reagent Refractory Regulation Resolution Ribosomes Role Saccharomyces cerevisiae Scanning Site Stimulus Stress Structure Time Translating Translation Initiation Translations Virus Diseases Yeasts base cofactor defined contribution design developmental disease eIF-4B experimental study extracellular flexibility helicase in vivo insight reconstitution response single molecule synthetic biology therapeutic development transcriptome

项目摘要

Regulated protein synthesis, or translation, is essential for life, and allows the cell to flexibly respond to external stimuli and stress. Conversely, dysregulated translation is a hallmark of diseases including cancer, viral infection, and developmental disorders. Translation is regulated principally through its initiation phase, where a crucial regulatory function of the initiation machinery is to ensure selection of the correct translation start site on messenger RNA. Failure to do so compromises the proteome by permitting synthesis of elongated, truncated, or nonsense proteins. In eukaryotes, start-site selection requires a directional search beginning at the 5’ end of the message. This search must move the megadalton ribosomal pre-initiation complex (PIC) efficiently through mRNA leader sequences that can span tens, hundreds, or even over a thousand nucleotides, and then halt this motion at exactly the correct start site. A linear “scanning” mechanism was first proposed over 40 years ago for this remarkable biophysical feat. However, fundamental properties of scanning have never been directly validated experimentally, and alternative mechanisms have been proposed. The importance of motion through the mRNA leader in translational control has also been brought into renewed sharp focus in recent years with the discovery that many mRNAs contain upstream open reading frames in their leaders that control translation of the main open reading frame; the scanning mechanism lies at the heart of how these are utilized. A critical barrier to progress is the remarkable molecular complexity and dynamism of the scanning machinery, whose numerous transient intermediates have made it challenging to characterize experimentally. Directly visualizing scanning in real time would allow many key unsolved questions to be addressed. Single-molecule methods are uniquely positioned to do this with the molecular resolution required to dissect mechanism. We have developed a single-molecule fluorescence assay for scanning of a reconstituted yeast PIC on full-length mRNAs. Here we will apply this assay to address the scanning mechanism. In Aim 1 we will directly determine the physical mechanism of motion in scanning, establishing the contributions of mRNA sequence and structure to the scanning rate. In Aim 2, we will elucidate how scanning directionality is established and maintained, focusing on the central translational helicase, eIF4A. We will distinguish between proposed mechanisms for how eIF4A transduces the chemical potential of ATP to bias scanning direction. In Aim 3, we will define the roles of pre- initiation complex components in scanning, with experiments that isolate their contribution to scanning specifically, rather than their aggregate functions throughout initiation. These studies will establish a physical- mechanistic model for scanning that will deepen understanding of translational control in health, and inform ongoing efforts to understand and reverse dysregulation in disease.

受调节的蛋白质合成或翻译对于生命至关重要，并允许细胞灵活地响应外部环境离线刺激和压力，失调的翻译是癌症、病毒感染等疾病的标志。翻译主要通过其起始阶段进行调节，这是一个关键的阶段。起始机制的调节功能是确保选择正确的翻译起始位点如果不这样做，就会导致延长的、截短的、在真核生物中，起始位点选择需要从 5' 端开始进行定向搜索。该搜索必须有效地移动兆道尔顿核糖体前起始复合物（PIC）。 mRNA 前导序列可以跨越数十个、数百个甚至超过一千个核苷酸，然后停止此过程 40 多年前首次提出了线性“扫描”机制。然而，扫描的基本特性从未被直接揭示。经过实验验证，并提出了替代机制。近年来，翻译控制领域的 mRNA 领导者也再次受到人们的关注。发现许多 mRNA 在其前导序列中含有控制翻译的上游开放阅读框主要开放阅读框的结构；扫描机制是如何利用这些内容的关键。进步的障碍是扫描机器显着的分子复杂性和活力，其大量的瞬态中间体使得直接可视化表征变得具有挑战性。实时扫描将能够解决许多未解决的关键问题。我们开发了解剖机制所需的分子分辨率，具有独特的优势。用于扫描全长 mRNA 上的重组酵母 PIC 的单分子荧光测定。将应用此分析来解决扫描机制。在目标 1 中，我们将直接确定物理机制。扫描中的运动机制，确定 mRNA 序列和结构对扫描的贡献在目标 2 中，我们将阐明如何建立和维持扫描方向性，重点是我们将区分中央翻译解旋酶 eIF4A 的机制。将 ATP 的化学势转换为偏向扫描方向在目标 3 中，我们将定义 pre- 的作用。在扫描中启动复杂的组件，并通过实验隔离它们对扫描的贡献具体来说，这些研究将建立一个物理-，而不是它们在整个启动过程中的聚合功能。扫描机制模型将加深对健康转化控制的理解，并提供信息持续努力了解和扭转疾病失调。