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

从一端到另一端：人类翻译起始的动态及其控制

• 批准号: 10543842
• 负责人: Christopher Paul Lapointe
• 金额: $ 6.44万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543842
• 关键词: Binding; Biochemical; Biological Assay; Biophysics; Cell physiology; Cells; Collaborations; Complement; Complex; Cryoelectron Microscopy; Data; Development; Dimensions; Disease; Embryo; Ensure; Event; Foundations; Future; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hour; Human; In Vitro; Kinetics; Length; Link; Malignant Neoplasms; Malignant neoplasm of lung; Mentors; Messenger RNA; Molecular; Molecular Conformation; Multiprotein Complexes; Mutation; Nervous System; Outcome; Phase; Postdoctoral Fellow; Process; Production; Protein Biosynthesis; Proteins; Regulation; Research; Research Personnel; Resolution; Ribosomes; Role; Site; Spectrum Analysis; Structure; Time; Training; Translation Initiation; Translational Repression; Translational Research; Translations; Viral Proteins; Virus Diseases; Work; base; developmental disease; eukaryotic initiation factor-5B; genetic regulatory protein; human disease; malignant neurologic neoplasms; nervous system disorder; particle; reconstitution; recruit; single molecule; structural biology; therapeutic development

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.

项目摘要 蛋白质合成是通过多步且高度调节的过程开始的，该过程用核糖体毒 在Messenger RNA（mRNA）上的起始位置。失控的丧失与人类疾病有关，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 局限性，一个重要的监管策略涉及招募到另一端的多蛋白质复合物 mRNA。招募的调节蛋白直接增强或抑制主动机械的组装 mRNA，从而调整蛋白质产生。基于翻译的许多分子机制 启动及其远程控制尚不清楚。当前的范例依赖于复合物的分析 稳定数分钟到几个小时，因为固有动力学挑战在批量解决方案中进行了稳定。在这里，我建立了 我最初的博士后研究，用于跟踪人类翻译起始机械，mRNA和调节 复合物在体外使用单分子光谱和纯化成分相互作用时，它们 补充结构分析。在AIM 1（K99）中，我关注核糖体亚基如何招募到 并加载到mRNA上，并确定这些里程标的事件如何由mRNA特征决定。 在AIM 2（K99）中，我研究了最终的启动步骤和活性蛋白质合成的过渡如何 由普遍保存的GTPase EIF5B协调和支配。在AIM 3（R00）中，我利用了所获得的 培训和专业知识，以检查如何通过mRNA的3'末端控制翻译启动 CCR4不复杂，是具有人类健康相关性的主要监管机构。正如我的初步数据所证明的那样，我的 策略将克服以前的障碍，以提供关键分子分支点的动态视图 基础翻译启动，揭示它们是如何针对对照的，并可能定义的分子碱基 疾病。在强大的合作和我的心理团队的帮助下，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，并增强 我的专业发展。当我过渡到 独立并继续我对翻译控制及其在人类疾病中如何出现的调查。