Dynamics of Eukaryotic Ribosomal Scanning

受调控的蛋白质合成或翻译是生命所必需的，并使细胞能够灵活地对外部环境做出反应 刺激和压力。相反，翻译失调是癌症、病毒感染等疾病的标志， 和发育障碍。翻译主要是通过其启动阶段进行管理的，在该阶段，关键的 启动机制的调节功能是确保选择正确的翻译起点 信使核糖核酸。如果做不到这一点，就会允许合成延长的、截断的、 或者是无意义的蛋白质。在真核生物中，起始点的选择需要从5‘端开始定向搜索 这条信息。这种搜索必须有效地将百万吨级的核糖体起始前复合体(PIC) 可以跨越数十个、数百个甚至一千多个核苷酸的信使核糖核酸前导序列，然后停止这种 在正确的起始点上移动。一种线性“扫描”机制在40多年前首次被提出，用于 这项非凡的生物物理壮举。然而，扫描的基本属性从来没有直接 经过实验验证，并提出了可供选择的机构。运动通过的重要性 近年来，在翻译控制方面的领先者也重新成为人们关注的焦点 发现许多mRNA在其前导中含有控制翻译的上游开放阅读框架 主要的开放阅读框架；扫描机制是如何利用这些的核心。一位批评者 阻碍进步的障碍是扫描机器惊人的分子复杂性和动力性，其 大量的瞬时中间体使实验表征变得具有挑战性。直接可视化 实时扫描将使许多关键的悬而未决的问题得到解决。单分子方法有 独一无二的定位，可以通过剖析机理所需的分子分辨率来实现这一点。我们已经开发出 单分子荧光法扫描重组酵母PIC全长mRNAs。在这里我们 将应用这种分析来解决扫描机制。在目标1中，我们将直接确定物理 扫描中的运动机制，建立mRNA序列和结构对细胞的贡献 扫描速度。在目标2中，我们将阐明如何建立和保持扫描方向性，重点是 中心翻译解旋酶eIF4A。我们将区分建议的eIF4A如何 将三磷酸腺苷的化学势转换为偏置扫描方向。在目标3中，我们将定义Pre- 在扫描中启动复杂成分，并通过实验分离它们对扫描的贡献 具体地说，而不是他们在整个印心过程中的聚合功能。这些研究将建立一个物理- 扫描的机械模型将加深对健康中的转换控制的理解，并提供 目前正在努力了解和扭转疾病中的调节失调。

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.