Dynamics of Eukaryotic Ribosomal Scanning

真核核糖体扫描动力学

• 批准号: 10034428
• 负责人: Sean E O'Leary
• 金额: $ 31.89万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 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/10034428
• 关键词: 5' 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

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生命至关重要，并使细胞灵活地响应外部 刺激和压力。相反，失调的翻译是疾病的标志，包括癌症，病毒感染， 和发展疾病。翻译主要通过其主动阶段进行调节，其中关键 主动机械的监管功能是确保选择正确的翻译起始站点 Messenger RNA。不这样做会通过允许伸长，截断， 或胡说八道的蛋白质。在真核生物中，开始站点的选择需要从5'结束开始的定向搜索 消息。该搜索必须通过有效地移动Megadalton核糖体预生效复合物（PIC） mRNA领导序列可以跨越数十万，数百甚至超过一千个核动肽，然后停止 在正确的开始站点上运动。 40年前首次提出了线性的“扫描”机制 这一非凡的生物物理壮举。但是，扫描的基本属性从未直接直接 经过实验验证，并提出了替代机制。运动的重要性 近年来，转化控制方面的mRNA领导者也已成为重点的重点 许多mRNA在其领导者中包含上游开放阅读框的发现，以控制翻译 主要的开放阅读框架；扫描机制是如何利用它们的核心。批判 进步的障碍是扫描机制的显着分子复杂性和动态性 许多瞬态中间体使实验表征表征挑战。直接可视化 实时扫描将允许许多关键的未解决问题解决。单分子方法是 独特的位置可以通过剖析机制所需的分子分辨率来执行此操作。我们已经发展了 单分子荧光测定法，用于扫描全长mRNA上的重构酵母菌图片。我们在这里 将应用此测定法以解决扫描机制。在AIM 1中，我们将直接确定物理 扫描中运动机理，建立mRNA序列和结构对 扫描率。在AIM 2中，我们将阐明如何建立和维护扫描方向性 中央翻译解旋酶EIF4A。我们将区分提出的EIF4A的机制 转导ATP的化学潜力到偏置扫描方向。在AIM 3中，我们将定义前的作用 扫描中的启动复合物成分，并通过隔离扫描的贡献的实验 具体而言，而不是整个计划中的总体功能。这些研究将建立一个身体 扫描的机械模型，将加深对健康转化控制的理解，并告知 持续了解和逆转疾病失调的努力。