The Conformational Dynamics of eIF5B During Ribosomal Subunit Joining

摘要 真核生物中的翻译启动需要十几个真核启动因子(EIF)，是一种 高度调控的过程，有助于建立蛋白质合成的保真度，并在细胞内直接作为靶点 压力和疾病。为了增加这个过程的复杂性，核糖体起始复合体(IC)经历了 EIF结合和活性的多重构象重排以及相关蛋白的失调 构象变化被认为是细胞压力和疾病的基础。尽管这些都很重要 翻译机器的功能的分子重排，目前的结构方法不能 解析从以下推断的EIF结合事件和IC结构重排的实时动力学 X射线结晶学和低温电子静态结构快照的比较 显微镜研究。这导致了我们对eIF结合和eIF-和核糖体如何结合的理解上的差距 结构重排相互配合，保证了翻译启动的保真度。具体地说， 大的核糖体亚基连接到IC的起始途径起到最终调节作用 小路上的检查站。这一步骤受翻译的鸟苷三磷酸酶(GTPase)eIF的调节 5B和EIF 1A。具体地说，5B加速了GTP和甲硫化启动子tRNA中的亚基连接步骤 (MET-tRNAi)依赖的方式，由1A促进。这导致了这样的问题：动态结构 5B的重排是其加速亚基连接的能力的基础？如果是这样，GTP和Met-tRNAi是如何 调制5B的动力学？同样，1A是如何调节5B的动态的，以及这样做是如何 调制调节5B识别Met-tRNAi、加速亚基连接和GTP的能力 亚基连接时的水解度？为了解决这些问题，我将使用高级单项- 分子Förster共振能量转移(SmFRET)技术及其在生物化学和生物物理学中的应用 表征亚基连接过程中5B、1A和IC的动态结构重排的方法， 并阐明了这些动力对翻译的机制和调节的贡献 入会仪式。这项提议的直接目标是剖析5B和5B的构象动力学和动力学 1A，展示了这些EIF如何将Met-tRNAi识别与亚基连接的加速GTP结合起来 被5B水解，以及在生产亚基连接后5B和1A的解离。

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.