Structural Dynamics of Translation

描述（由申请人提供）：核糖体在所有生物体中合成蛋白质。尽管蛋白质合成的研究取得了重大进展，但翻译的许多分子细节尚不清楚，核糖体的研究仍然是结构生物学中最大的挑战之一。蛋白质合成是一个动态过程，核糖体沿着mRNA移动，trna在核糖体内易位。该项目的长期目标是阐明核糖体和核糖体配体在翻译基础上的构象转变。该提案侧重于核糖体沿mRNA运动的机制和核糖体循环的分子机制，即在蛋白质合成终止后核糖体分解成亚基。细菌中的这两个过程都涉及蛋白质因子EF-G（真核生物中的EF-2）。易位和再循环的分子细节尚不清楚。研究表明，核糖体沿mRNA的易位需要核糖体亚基之间的棘轮状旋转，而亚基之间的旋转是热驱动的，并且是自发发生的。该提案的具体目标1是探索核糖体作为布朗棘轮机器的机械特性，其自发的亚基间旋转可以通过配体结合纠正为易位。我们将使用抗生素作为工具来研究结合位点与大核糖体亚基上a位点重叠的抗生素是否能诱导核糖体易位。特异性目标2将解决核糖体易位的天然催化剂EF-G如何将自发的亚基间运动纠正为核糖体的单向易位的问题。EF-G的扩展结构域IV已被证明与易位后核糖体的A位点结合。据推测，扩展结构域IV相对于A位点和EF-G其余部分的移动对易位至关重要。我们将在单分子和停流动力学测量中使用f¿rster共振能量转移（FRET），通过跟踪EF-G在易位过程中的第四域运动来验证这一假设。Specific Aim 1和2的实验将阐明易位的机制，并有助于理解分子机器（如DNA/RNA聚合酶和解旋酶）如何沿着核酸移动。EF-G还参与由EF-G和核糖体再循环因子（RRF）共同作用诱导的核糖体再循环。具体目标3是研究核糖体、RRF和EF-G的构象变化与使用FRET的核糖体回收有关。提出的实验将揭示核糖体循环过程中的结构转变序列，并有助于了解其分子机制。我们的研究将阐明蛋白质合成的基本步骤，从而回答基本的生物学问题。

DESCRIPTION (provided by applicant): The ribosome synthesizes proteins in all living organisms. Despite significant progress in the research of protein synthesis, many molecular details of translation are unknown and the study of the ribosome remains one of the biggest challenges in structural biology. Protein synthesis is a dynamic process, during which the ribosome moves along mRNA and tRNAs are translocated inside the ribosome. The long-term goal of the proposed project is to elucidate conformational transitions of the ribosome and ribosomal ligands underlying translation. The proposal is focused on the mechanics of ribosome movement along mRNA and the molecular mechanism of ribosome recycling, i.e., ribosome disassembly into subunits after termination of protein synthesis. Both of these processes in bacteria involve protein factor EF-G (EF-2 in eukaryotes). Molecular details of translocation and recycling are poorly understood. It has been shown that ribosome translocation along mRNA requires ratchet-like rotation between ribosomal subunits and that intersubunit rotation is thermally driven and occurs spontaneously. The Specific Aim 1 of the proposal is to probe mechanistic properties of the ribosome as a Brownian ratchet machine whose spontaneous intersubunit rotation can be rectified into translocation by ligand binding. We will use antibiotic as a tool to examine whether antibiotics whose binding sites overlap with the A site on the large ribosomal subunit can induce ribosome translocation. The Specific Aim 2 will address the question of how the natural catalyst of ribosomal translocation, EF-G rectifies spontaneous intersubunit movements into unidirectional translocation of the ribosome. The extended domain IV of EF-G has been shown to bind to the A site of the post-translocation ribosome. It has been hypothesized that movement of the extended domain IV relative to the A site and the rest of EF-G is critical for translocation. We are going to test this hypothesis by following movements of domain IV of EF-G during translocation using F¿rster resonance energy transfer (FRET) in single-molecule and stopped-flow kinetic measurements. The experiments of Specific Aim 1 and 2 will elucidate the mechanism of translocation and contribute to understanding of how molecular machines, such as DNA/RNA polymerases and helicases, move along nucleic acids. EF-G is also involved in ribosome recycling that is induced by the concerted action of EF-G and ribosome recycling factor (RRF). The Specific Aim 3 is to study conformational changes of the ribosome, RRF and EF-G associated with ribosome recycling using FRET. Proposed experiments will reveal the sequence of structural transitions during ribosome recycling and help to understand its molecular mechanism. Our studies will illuminate essential steps of protein synthesis and thus answer fundamental biological questions. PUBLIC HEALTH RELEVANCE: Search for new classes of antibiotics becomes increasingly important because of the growing problem of pathogen resistance to existing antibacterials. Many widely-used antibiotics inhibit protein synthesis in the cell and target bacterial ribosomes. The proposed studies aim to reveal molecular details of protein synthesis, and will have a significant impact on medical research by contributing to our understanding of the mechanism of antibiotic action, drug resistance, and development of new drugs.