Dynamics of Eukaryotic Ribosomal Scanning

蛋白质的合成或翻译是生命所必需的，它允许细胞灵活地对外界环境做出反应。 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， 和发育障碍。翻译主要是通过其初始阶段进行调节的，在此阶段， 起始机制的调节功能是确保选择正确的翻译起始位点， 信使RNA。如果不能这样做，蛋白质组就会受到损害， 或无义蛋白质。在真核生物中，起始位点的选择需要从5'末端开始的定向搜索。 留言加载这种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，然后停止这一过程。 在正确的起始点运动。线性“扫描”机制在40多年前首次提出， 这一非凡的生物物理学壮举然而，扫描的基本属性从未直接 实验验证，并提出了替代机制。运动的重要性 近年来，翻译控制中mRNA前导序列也重新成为人们关注的焦点， 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 主要开放式阅读框架的核心;扫描机制是如何利用这些框架的核心。一个关键 进步的障碍是扫描机器的显着分子复杂性和动态性， 许多瞬时中间体使得实验表征具有挑战性。直接可视化 真实的扫描将允许解决许多关键的未解决的问题。单分子方法是 独特的定位，以做到这一点与所需的分子分辨率解剖机制。我们已经开发 单分子荧光分析用于扫描重组酵母PIC上的全长mRNA。这里我们 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 在扫描中的运动机制，建立mRNA序列和结构的贡献， 扫描速率在目标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eIF 4A。我们将区分eIF 4A如何 转换ATP的化学势以偏置扫描方向。在目标3中，我们将定义前- 启动复杂的组件在扫描，与实验，隔离他们的贡献扫描 具体来说，而不是他们在整个启动过程中的总功能。这些研究将建立一个物理- 扫描的机械模型，将加深对健康翻译控制的理解， 正在努力了解和扭转疾病的失调。

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.