The structural dynamics of translation initiation

翻译起始的结构动力学

• 批准号: 8280256
• 负责人: Ruben L Gonzalez
• 金额: $ 5.99万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280256
• 关键词: Active Sites; Address; Affect; Antibiotics; Anticodon; Binding; Biochemical; Biological Process; Cells; Codon Nucleotides; Complex; Coupling; Data; Dependence; Development; Disease; Docking; Enzymes; Equilibrium; Event; Fluorescence; Gene Expression; Health; Heterogeneity; Human; Individual; Initiator Codon; Initiator tRNA; Kinetics; Label; Life; Link; Literature; Malignant Neoplasms; Messenger RNA; Methods; Microscopic; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Pathway interactions; Peptide Initiation Factors; Peptidyltransferase; Phase; Population; Positioning Attribute; Process; Prokaryotic Initiation Factor-2; Protein Biosynthesis; Proteins; Reaction; Regulation; Reporting; Resolution; Ribonucleoproteins; Ribosomes; Role; Series; Staging; Structure; System; Techniques; Technology; Testing; Time; Transfer RNA; Translating; Translation Initiation; Translation Process; Translations; Viral; base; pathogen; research study; response; single molecule; single-molecule FRET; small molecule; structural biology; tool; tumorigenesis

DESCRIPTION (provided by applicant): The ribosome, a large ribonucleoprotein enzyme, is universally responsible for translating messenger RNAs (mRNAs) into the encoded protein products. This process is among one of the most fundamental and highly regulated in all living things. Recent advances in the structural biology of protein synthesis have provided atomic resolution structures of the ribosome as well as lower-resolution snapshots of ribosomal complexes trapped in the process of translation. What is currently lacking from mechanistic models of ribosome function is a description of the kinetics governing transitions from one conformational state of the ribosome to the next. Although difficult, and often impossible, to study precisely using bulk biochemical methods, these conformational dynamics have been shown to be of prime importance in translation. The initiation phase of protein synthesis is the focal point for the translational control of gene expression. As such, the initiation pathway serves as a very effective target for small molecule antibiotics, human viral pathogens, and deregulation of initiation is increasingly causally linked to tumorigenesis. The initiation reaction is an amazingly dynamic process, involving the interaction of numerous translation initiation factors (IFs) with the ribosome in a highly-coordinated and specific series of molecular events. We hypothesize that IFs regulate the initiation pathway by precisely altering the stabilities of dynamically heterogeneous conformational intermediates of the initiation machinery. To address this dynamic conformational heterogeneity, we will use single-molecule fluorescence resonance energy transfer (smFRET). smFRET provides a unique tool for characterizing the conformational dynamics of individual molecules, eliminating the population averaging inherent in ensemble studies and revealing the dynamic heterogeneity of the system. These data will help elucidate the basic mechanism of translation initiation, providing crucial kinetic information that has heretofore remained inaccessible in bulk studies. Specifically, we will use these techniques to (1) investigate the dynamics of initiation factor 2 (IF2) and initiator transfer RNA (tRNAi) that regulate tRNAi selection during initiation, (2) determine how coupling of ribosome and tRNAi conformational dynamics control the fidelity of tRNAi and start codon selection, and (3) establish the currently unknown mechanism through which initiation factor 3 (IF3) acts to proofread the fidelity of the initiation reaction. Our ability to correlate the kinetics of critical conformational changes with fundamental biochemical steps in the initiation pathway will aid the development of a complete mechanistic model for this universal and biomedically relevant biological process. PUBLIC HEALTH RELEVANCE: Protein synthesis, catalyzed in all cells by an enzyme called the ribosome, is an important focal point for the control of gene expression. Loss of control over the initiation step of protein synthesis is induced by antibiotic drugs, is exploited by human viral pathogens, and is implicated in cancer. This proposal uses state-of-the-art microscopic technologies to addresses fundamental aspects of initiation that hold great promise towards revealing how this step in gene expression is controlled and how that control is exploited in disease.

描述（由申请人提供）：核糖体是一种大型核糖核蛋白酶，普遍负责将信使 RNA (mRNA) 翻译成编码的蛋白质产物。这个过程是所有生物中最基本、最受监管的过程之一。蛋白质合成结构生物学的最新进展提供了核糖体的原子分辨率结构以及翻译过程中捕获的核糖体复合物的较低分辨率快照。目前核糖体功能的机械模型缺乏的是对控制核糖体从一种构象状态到下一种构象状态转变的动力学的描述。尽管使用大量生化方法进行精确研究很困难，而且通常是不可能的，但这些构象动力学已被证明在翻译中至关重要。蛋白质合成的起始阶段是基因表达翻译控制的焦点。因此，起始途径是小分子抗生素、人类病毒病原体的非常有效的靶标，并且起始的失调与肿瘤发生越来越有因果关系。起始反应是一个令人惊奇的动态过程，涉及众多翻译起始因子 (IF) 与核糖体在高度协调和特定的一系列分子事件中的相互作用。我们假设 IF 通过精确改变起始机制的动态异质构象中间体的稳定性来调节起始途径。为了解决这种动态构象异质性，我们将使用单分子荧光共振能量转移（smFRET）。 smFRET 提供了一种独特的工具来表征单个分子的构象动力学，消除了整体研究中固有的群体平均并揭示了系统的动态异质性。这些数据将有助于阐明翻译起始的基本机制，提供迄今为止在批量研究中无法获得的关键动力学信息。具体来说，我们将使用这些技术来 (1) 研究起始因子 2 (IF2) 和起始转移 RNA (tRNAi) 的动力学，这些动力学在起始过程中调节 tRNAi 选择，(2) 确定核糖体和 tRNAi 构象动力学的耦合如何控制保真度tRNAi 和起始密码子选择，以及 (3) 建立目前未知的机制，通过该机制起始因子 3 (IF3) 可以发挥作用来校对起始反应的保真度。我们将关键构象变化的动力学与起始途径中的基本生化步骤关联起来的能力将有助于为这种普遍且与生物医学相关的生物过程开发完整的机制模型。公共健康相关性：所有细胞中由核糖体酶催化的蛋白质合成是控制基因表达的重要焦点。蛋白质合成起始步骤的失控是由抗生素药物引起的，被人类病毒病原体利用，并且与癌症有关。该提案使用最先进的显微技术来解决启动的基本方面，这对于揭示基因表达中的这一步骤如何被控制以及如何在疾病中利用这种控制具有很大的希望。