The Conformational Dynamics of eIF5B During Ribosomal Subunit Joining

核糖体亚基连接过程中 eIF5B 的构象动力学

• 批准号: 10295756
• 负责人: Erik W Hartwick
• 金额: $ 6.31万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-11-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10295756
• 关键词: Acceleration; Acylation; Address; Behavior; Binding; Biochemical; C-terminal; Cellular Stress; Complex; Disease; Dissociation; Electron Microscopy; Ensure; Eukaryota; Eukaryotic Initiation Factors; Event; Fluorescence Resonance Energy Transfer; Goals; Guanosine; Guanosine Triphosphate; Hydrolysis; In Vitro; Initiator tRNA; Investigation; Kinetics; Label; Link; Mediating; Messenger RNA; Methods; Molecular; Molecular Conformation; Nucleotides; Pathway interactions; Peptide Initiation Factors; Process; Protein Biosynthesis; Regulation; Reporting; Ribosomes; Roentgen Rays; Signal Transduction; System; Tail; Techniques; Time; Translating; Translation Initiation; Translations; Yeasts; biophysical techniques; cryogenics; eukaryotic initiation factor-5B; experimental study; fluorophore; insight; loss of function; loss of function mutation; molecular rearrangement; mutant; prevent; single molecule; single-molecule FRET

ABSTRACT Translation initiation in eukaryotes, which requires over a dozen eukaryotic initiation factors (eIFs), is a highly regulated process that helps establish the fidelity of protein synthesis and is directly targeted during cellular stress and disease. To add to the complexity of this process, ribosomal initiation complexes (ICs) undergo multiple conformational rearrangements upon eIF binding and activity, and dysregulation of the associated conformational changes are thought to underlie cellular stress and disease. Despite the importance of these molecular rearrangements for the function of the translation machinery, current structural approaches cannot resolve the real-time dynamics of the eIF binding events and IC structural rearrangements that are inferred from comparisons of the static structural snapshots provided by X-ray crystallographic and cryogenic electron microscopy studies. This presents a gap in our understanding of how eIF binding and eIF- and ribosome structural rearrangements cooperatively ensure the fidelity of translation initiation. Specifically, the last step in the initiation pathway, in which the large ribosomal subunit joins to the IC, functions as a final regulatory checkpoint in the pathway. This step is regulated by the translational guanosine triphosphatase (GTPase) eIF 5B and eIF 1A. Specifically, 5B accelerates the subunit joining step in a GTP- and methionylated initiator tRNA (Met-tRNAi)-dependent manner that is facilitated by 1A. This leads to questions such as: Do dynamic structural rearrangements of 5B underlie its ability to accelerate subunit joining? If so, how do GTP and Met-tRNAi modulate the dynamics of 5B? Likewise, how does 1A modulate the dynamics of 5B and how does such modulation regulate the ability of 5B to recognize Met-tRNAi, accelerate subunit joining, and undergo GTP hydrolysis upon subunit joining? To address these questions, I will use a combination of advanced single- molecule Förster Resonance Energy Transfer (smFRET) techniques and ensemble biochemical and biophysical approaches to characterize the dynamic structural rearrangements of 5B, 1A, and the IC during subunit joining, and elucidate the contributions that these dynamics make to the mechanism and regulation of translation initiation. The immediate goal of this proposal is to dissect the conformational dynamics and kinetics of 5B and 1A, demonstrating how these eIFs couple Met-tRNAi recognition to acceleration of subunit joining, GTP hydrolysis by 5B, and dissociation of 5B and 1A following productive subunit joining.

摘要 真核生物中的翻译起始需要十几个真核生物起始因子（eIF）， 一个高度调节的过程，有助于建立蛋白质合成的保真度，并在细胞生长过程中直接靶向 压力和疾病。为了增加这一过程的复杂性，核糖体起始复合物（IC）经历了 eIF结合和活性后的多重构象重排，以及相关的 构象变化被认为是细胞应激和疾病的基础。尽管这些重要性 分子重排的翻译机制的功能，目前的结构方法不能 解析eIF结合事件和IC结构重排的实时动态， X射线晶体学和低温电子学提供的静态结构快照的比较 显微镜研究。这在我们理解eIF结合以及eIF和核糖体 结构重排协同确保翻译起始的保真度。具体来说，最后一步 起始途径，其中大的核糖体亚基连接到IC，作为一个最终的调节， 检查站在路上。这一步由翻译的鸟苷三磷酸酶（GTdR）eIF调节 5 B和eIF 1A。具体而言，5 B加速GTP-和甲硫氨酰化起始tRNA中的亚基连接步骤 （Met-tRNAi）依赖性方式，其由1A促进。这就引出了如下问题：动态结构 5 B重排的基础，其能力，以加速亚基加入？如果是这样，GTP和Met-tRNAi 调节5 B的动态？同样，1A如何调节5 B的动力学， 调节调节5 B识别Met-tRNAi的能力，加速亚基连接，并进行GTP 亚基连接后水解？为了解决这些问题，我将使用先进的单- 分子Förster共振能量转移（smFRET）技术和整体生物化学和生物物理 表征5 B、1A和IC在亚基连接期间的动态结构重排的方法， 并阐明这些动力学对翻译机制和翻译调控的贡献 入会仪式该提议的直接目标是剖析5 B的构象动力学和动力学， 图1A，展示了这些eIF如何将Met-tRNAi识别与亚基连接、GTP 通过5 B的水解，以及在生产性亚基连接后5 B和1A的解离。