Dynamics of eukaryotic translation initiation and its control

项目概要（30行） 真核生物中翻译的起始是限速的并且受到高度调节。过程 涉及将40 S亚基引导至蛋白质的5'非翻译区（5' UTR）的蛋白质因子的得分。 mRNA，随后定向扫描到第一个起始密码子，然后是60 S亚基 连接并过渡到伸长。这一过程的机制基础尚不清楚。 在之前的资助期间，我们开发了单分子方法，以提供实时 酵母翻译起始的动力学观点。在这里，我们建立在我们的方法论 过去资助期间的发展，以提供结构和动态的综合观点， 在酵母和人类中是如何开始的。在目标1中，我们关注40 S亚基的制备 然后连接mRNA的5'端，并利用我们的标记因子来理解 eIF 4F复合物通过与mRNA协作来指导这一过程的动力学， 解旋酶;然后，我们将探讨定向扫描的起始密码子，并描绘的作用 5' UTR长度序列和结构以及3'末端蛋白质和RNA如何调节 过程目的2研究了起始密码子识别是如何通过60 S亚基发生的 连接和向伸长的过渡。我们确定构象/组成信号， 起始密码子识别，以及为什么eIF 5 B诱导的延伸过渡缓慢，以及是否 引发因素在伸长期间持续存在。我们将把慢事件与已知的体内 测量和探测扫描中排队的后果。在目标3中，我们研究 替代起始途径，如泄漏扫描，同源起始位点附近和上游 开放式阅读框架翻译、终止和重新启动。为了所有的目标，我们合并动态 通过cryoEM对中间体进行结构表征的生物化学研究 由我们的单分子观测引导。这项研究得到了强有力的支持。 在各种系统上的合作，以支持生物物理和结构分析，试剂 制备和体内相关性。这项提案的结果将提供深入和广泛的 鉴于mRNA序列和结构的相互作用以及翻译起始途径， 为理解疾病中的翻译控制及其错误调节提供了基础。

PROJECT SUMMARY (30 lines) Initiation of translation in eukaryotic organisms is rate limiting and highly regulated. The process involves a score of protein factors guiding 40S subunits to the 5’ untranslated region (5’UTR) of an mRNA, and subsequent directional scanning to the first start codon, followed by 60S subunit joining and transition to elongation. The mechanistic basis for this process remains unclear. During the prior funding period we developed single-molecule approaches to provide a real-time dynamic perspective to yeast translation initiation. Here we build on our methodological developments over the past funding period to provide combined structural and dynamic view of how initiation occurs in yeast and humans. In Aim 1 we focus on how 40S subunits are prepared and then join the 5’ end of an mRNA and leverage our labeled factors to understand the dynamics by which the eIF4F complex guides this process in collaboration with mRNA and helicases; we then will explore the directional scanning to the start codon and delineate the role of 5’ UTR length sequence and structure and how 3’ terminal proteins and RNA modulate the process. In Aim 2, we investigate how start codon recognition occurs through 60S subunit joining and the transition to elongation. We determine the conformation/composition signals in start codon recognition, and why eIF5B induced transition to elongation is slow, and whether initiation factors linger during elongation. We will correlate slow events with known in vivo measurements and probe the consequences of queuing in scanning. In Aim 3, we investigate alternative initiation pathways, such as leaky scanning, near cognate start sites and upstream open reading frame translation, termination and re-initiation. For all aims, we merge dynamic and biochemical investigations with structural characterization of intermediates by cryoEM guided by our single-molecule observations. The proposed research is buttressed by strong collaborations on the various systems to support biophysical and structural analysis, reagent preparation, and in vivo correlation. The results of this proposal will provide deep and broad view of the interplay of mRNA sequence and structure and the pathways of translation initiation, providing a foundation to understand translational control and its mis-regulation in disease.