From one end to the other: dynamics of human translation initiation and its control

项目摘要 蛋白质合成开始于一个多步骤和高度调节的过程，最终以核糖体平衡 在信使RNA（mRNA）上的起始位点。失控广泛涉及人类疾病，包括 癌症、发育障碍、神经系统疾病和病毒感染。由于翻译启动是速率 限制，一个重要的调控策略涉及多蛋白复合物招募到相反的末端， mRNA。募集的调节蛋白直接增强或抑制起始机制在细胞膜上的组装。 mRNA，从而上调或下调蛋白质的产生。翻译的许多分子机制 其启动和远程控制仍不清楚。当前的范式依赖于对复合物的分析， 随着本体溶液中固有动力学挑战的临近，可稳定数分钟至数小时。在这里，我建立了 我最初的博士后研究是跟踪人类翻译起始机制，mRNA和调控机制， 复合物，因为他们相互作用，使用单分子光谱和纯化的成分在体外，我 补充结构分析。在目标1（K99）中，我专注于核糖体亚基是如何被募集到 并加载到mRNA上，并确定这些标志性起始事件如何由mRNA特征决定。 在目标2（K99）中，我研究了最终的起始步骤和向活性蛋白质合成的过渡是如何进行的。 由一个普遍保守的GT3，eIF 5 B协调和管理。在目标3（R 00）中，我利用获得的 培训和专业知识，以检查翻译起始是如何通过mRNA的3 '端控制的， CCR 4-不复杂，与人类健康相关的主要调节剂。正如我的初步数据显示， 战略将克服以前的障碍，提供关键分子分支点的动态视图， 作为翻译起始的基础，揭示它们是如何被靶向控制的，并可能定义翻译起始的分子基础。 疾病在强有力的合作和我的指导团队的帮助下，拟议的研究和培训计划将 为我提供结构生物学和生物物理学的新概念和实验专业知识，并提高 我的专业发展。总之，这份提案将成为我过渡到 独立性和继续我的调查翻译控制和它是如何出错的人类疾病。

Project Summary Protein synthesis begins via a multi-step and highly-regulated process that culminates with a ribosome poised at a start site on a messenger RNA (mRNA). Loss of control is broadly implicated in human disease, including cancers, developmental disorders, neurological diseases, and viral infections. Since translation initiation is rate limiting, an important regulatory strategy involves multi-protein complexes recruited to the opposite end of the mRNA. Recruited regulatory proteins directly enhance or inhibit assembly of the initiation machinery on the mRNA, thereby tuning protein production up or down. Many molecular mechanisms that underlie translation initiation and its long-range control remain unclear. Current paradigms rely on analyses of complexes that are stable for minutes to hours, as the intrinsic dynamics challenge approaches in bulk solutions. Here, I build off my initial postdoctoral research to track the human translation initiation machinery, mRNA, and regulatory complexes as they interact using single-molecule spectroscopy and purified components in vitro, which I complement with structural analyses. In Aim 1 (K99), I focus on how the ribosomal subunits are recruited to and load onto an mRNA, and determine how these landmark initiation events are dictated by mRNA features. In Aim 2 (K99), I examine how the final initiation steps and the transition into active protein synthesis are coordinated and governed by a universally-conserved GTPase, eIF5B. In Aim 3 (R00), I leverage the obtained training and expertise to examine how translation initiation is controlled via the 3’-end of the mRNA by the CCR4-NOT complex, a major regulator with human-health relevance. As my preliminary data demonstrate, my strategy will overcome previous roadblocks to provide a dynamic view of key molecular branchpoints that underlie translation initiation, reveal how they are targeted for control, and may define molecular bases of disease. Aided by strong collaborations and my mentoring team, the proposed research and training plan will provide me with new conceptual and experimental expertise in structural biology and biophysics and enhance my professional development. Together, this proposal will serve as a strong foundation as I transition into independence and continue my investigation of translational control and how it goes awry in human disease.