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、最受控制的过程之一。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。目前缺乏核糖体功能的机制模型是对核糖体从一种构象状态到另一种构象状态转变的动力学描述。虽然很难，而且往往不可能，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翻译的首要重要性。蛋白质合成的起始阶段是基因表达翻译控制的重点。因此，起始途径是小分子抗生素、人类病毒病原体的一个非常有效的靶标，而起始途径的放松与肿瘤发生的因果关系越来越密切。起始反应是一个惊人的动态过程，涉及许多翻译起始因子（IFs）与核糖体在一系列高度协调和特异性的分子事件中相互作用。我们假设干扰素通过精确改变启动机制中动态异质构象中间体的稳定性来调节启动途径。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总体平均，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，提供迄今为止在大量研究中仍然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）和启动器转移RNA （tRNAi）在起始过程中调控tRNAi选择的动力学，(2)确定核糖体和tRNAi构象动力学的耦合如何控制tRNAi的保真度和启动密码子选择，以及(3)建立目前未知的机制，通过启动因子3 （IF3）的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联系起来的能力将有助于为这一普遍和生物医学相关的生物过程建立完整的机制模型。

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.