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: 10877449
• 负责人: Christopher Paul Lapointe
• 金额: $ 24.9万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 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 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10877449
• 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 蛋白质合成通过一个多步骤和高度调控的过程开始，最终形成稳定的核糖体。 在信使RNA(信使RNA)的起始点。失控被广泛认为与人类疾病有关，包括 癌症、发育障碍、神经疾病和病毒感染。因为翻译启动是有费率的 限制，一个重要的调控策略涉及到招募到细胞另一端的多蛋白复合体 MRNA.招募的调节蛋白直接增强或抑制启动机制在 MRNA，从而调节蛋白质产量的增加或减少。翻译背后的许多分子机制 它的启动和远程控制仍不清楚。当前的范式依赖于对复杂结构的分析，这些复杂结构 随着批量解决方案中内在动态挑战的临近，稳定可持续数分钟至数小时。在这里，我在 我最初的博士后研究是跟踪人类翻译启动机制、信使核糖核酸和调控 使用单分子光谱学和体外纯化成分相互作用的化合物，这是 与结构分析相补充。在目标1(K99)中，我重点介绍核糖体亚基是如何被招募到 并加载到信使核糖核酸上，并确定这些里程碑式的启动事件如何由信使核糖核酸特征决定。 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GTP酶eIF5B协调和管理。在目标3(R00)中，我利用获得的 培训和专业知识，以研究翻译启动是如何通过信使核糖核酸的3‘端控制的 CCR4-不复杂，与人类健康相关的主要调节器。正如我的初步数据显示的那样，我的 战略将克服以前的障碍，提供关键分子分支点的动态视图， 翻译启动的基础，揭示它们是如何被控制的，并可能定义 疾病。在强大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方面的新概念和实验专业知识，并增强 我的职业发展。总而言之，这项提议将成为我过渡到 独立并继续我对翻译控制以及它在人类疾病中如何出错的研究。