The structural dynamics of translation initiation

描述(申请人提供)：核糖体是一种大的核糖核蛋白酶，普遍负责将信使RNA(MRNAs)翻译成编码的蛋白质产品。这一过程是所有生物中最基本、最严格管制的过程之一。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结构以及在翻译过程中捕获的核糖体复合体的低分辨率快照。目前核糖体功能的机制模型所缺乏的是对控制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。尽管使用大量生化方法精确研究这些构象动力学很困难，而且常常是不可能的，但这些构象动力学已经被证明在翻译中是最重要的。蛋白质合成的起始阶段是基因表达的翻译调控的焦点。因此，启动途径是小分子抗生素、人类病毒病原体非常有效的靶点，而启动的解除管制与肿瘤的发生越来越有因果关系。引发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通过精确地改变启动机制的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，消除了系综研究中固有的布居平均化，并揭示了系统的动态异质性。这些数据将有助于阐明翻译启动的基本机制，提供到目前为止在大规模研究中仍无法获得的关键动力学信息。具体地说，我们将使用这些技术来(1)研究在启动过程中调节tRNAi选择的启动因子2(IF2)和启动子转移RNA(TRNAi)的动力学，(2)确定核糖体和tRNAi构象动力学耦合如何控制tRNAi的保真度和开始密码子选择，以及(3)建立目前未知的启动因子3(IF3)作用于校对启动反应保真度的机制。我们能够将关键构象变化的动力学与启动途径中的基本生化步骤相关联，这将有助于为这一普遍的和生物医学相关的生物过程发展一个完整的机制模型。

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.