Structural Dynamics of Translation

翻译的结构动力学

• 批准号: 8442913
• 负责人: Dmitri Ermolenko
• 金额: $ 28.33万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8442913
• 关键词: Address; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biological; Catalysis; Cell physiology; Cells; Chloramphenicol; Coupled; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Dissociation; Drug resistance; EWS/FLI 1 Type 2 gene; Energy Transfer; Eukaryota; GTP Binding; Genetic; Goals; Guanosine Triphosphate; Homologous Gene; Hybrids; Hydrolysis; Kinetics; Knowledge; Laboratories; Life; Ligand Binding; Ligands; Measurement; Mechanics; Medical Research; Messenger RNA; Methods; Microscopy; Molecular; Molecular Machines; Molecular Motors; Movement; Nucleic Acids; Organism; Peptide Elongation Factor G; Pharmaceutical Preparations; Play; Process; Property; Protein Biosynthesis; Proteins; RNA; RNA Helicase; Recycling; Relative (related person); Research; Resistance; Resistance development; Rest; Ribosomes; Role; Rotation; Site; Sparsomycin; Structure; Techniques; Testing; Time; Transfer RNA; Translating; Translations; Work; catalyst; conformational conversion; design; helicase; pathogen; prevent; research study; ribosome releasing factor; single molecule; structural biology; tool

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.

描述（由适用提供）：所有生物体中的核糖体合成蛋白。尽管在蛋白质合成研究方面取得了重大进展，但许多翻译的分子细节尚不清楚，对核糖体的研究仍然是结构生物学的最大挑战之一。蛋白质合成是一个动态过程，在此过程中，核糖体沿mRNA和TRNA移动在核糖体内。拟议项目的长期目标是阐明核糖体和核糖体配体的构象转变。该建议集中于沿mRNA的核糖体运动的机理以及核糖体回收的分子机理，即核糖体在终止蛋白质合成后将其分解为亚基。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）。易位和回收的分子细节知之甚少。已经表明，沿mRNA的核糖体易位需要核糖体亚基之间的棘轮样旋转，并且亚基间的旋转是热驱动的，并且是由赞助的。该提案的具体目的1是将核糖体的机械特性探测为布朗棘轮机，其赞助的subersunit旋转可以通过配体结合将其纠正为易位。我们将使用抗生素作为一种工具来检查其结合位点与大核糖体亚基上A位点重叠的抗生素是否可以诱导核糖体易位。具体的目标2将解决核糖体易位的自然催化剂如何纠正赞成的亚基间运动中核糖体的单向易位。 EF-G的扩展域IV已被证明与跨化核糖体的A位点结合。已经假设，扩展域IV相对于A位点的运动和EF-G的其余部分对于易位至关重要。我们将通过在单分子和停止流动动力学测量中使用F rster共振能量转移（FRET）在转运过程中使用EF-G的域IV运动来检验这一假设。特定目标1和2的实验将阐明翻译机制，并有助于理解分子机器（例如DNA/RNA聚合酶和解旋酶）如何沿核酸移动。 EF-G还参与了核糖体回收，这是由EF-G和核糖体回收因子（RRF）的一致作用引起的。具体目的3是研究核糖体，RRF和EF-G的会议变化与使用FRET相关的核糖体回收。提出的实验将揭示核糖体回收过程中结构过渡的序列，并有助于理解其分子机制。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。