The structural dynamics of translation initiation

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和最受高度调节的过程之一。蛋白质合成的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在翻译过程中捕获的核糖体复合物的低分辨率快照。目前缺乏的是从核糖体功能的机制模型是一个动力学的描述，从一个核糖体的构象状态到下一个的过渡。虽然很难，而且往往是不可能的，精确地研究使用散装生化方法，这些构象动力学已被证明是最重要的翻译。蛋白质合成的起始阶段是基因表达的翻译控制的焦点。因此，起始途径作为小分子抗生素、人类病毒病原体的非常有效的靶标，并且起始的失调越来越多地与肿瘤发生因果相关。起始反应是一个惊人的动态过程，涉及许多翻译起始因子（IF）与核糖体在一系列高度协调和特定的分子事件中的相互作用。我们假设，IFs调控的起始途径，精确地改变稳定性的动态异构构象中间体的起始机制。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为表征单个分子的构象动力学提供了独特的工具，消除了系综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，提供重要的动力学信息，迄今为止仍然无法在批量研究。具体而言，我们将使用这些技术来（1）研究在起始期间调节tRNAi选择的起始因子2（IF2）和起始转移RNA（tRNAi）的动力学，（2）确定核糖体和tRNAi构象动力学的偶联如何控制tRNAi和起始密码子选择的保真度，以及（3）建立目前未知的机制，通过该机制起始因子3（IF3）起作用以校正起始反应的保真度。我们的能力，相关的关键构象变化的动力学与基本的生化步骤的起始途径将有助于发展一个完整的机械模型，这个普遍的和生物医学相关的生物过程。公共卫生相关性：蛋白质的合成，在所有细胞中由一种称为核糖体的酶催化，是控制基因表达的重要焦点。对蛋白质合成起始步骤的控制丧失由抗生素药物诱导，被人类病毒病原体利用，并与癌症有关。该提案使用最先进的显微技术来解决启动的基本方面，这些方面对揭示基因表达中的这一步骤是如何控制的以及这种控制如何在疾病中被利用具有很大的希望。

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.