The Structural Dynamics of Translation Initiation

项目摘要 双亚基核糖体在mRNA起始密码子处组装， 启动蛋白质合成是基因表达的最基本和高度调节的步骤之一。作为 这种起始作用是许多小分子抗生素和细胞病原体的靶点。此外，委员会认为， 起始的失调与人类的病毒感染和肿瘤发生有因果关系。鉴于这一切， 引发的分子机制的研究为鉴定和表征 可以作为下一代抗生素和其他小分子抗生素开发的目标的机械步骤， 分子、抗病毒和抗癌药物，其通过调节翻译起始起作用。 尽管他们的承诺，分子医学，机制的研究启动仍然令人难以置信的 挑战性这主要是因为启蒙是一个非常动态的、多步骤的过程， 通过大量难以观察和表征的短暂中间状态 使用传统的方法。在启动过程中，一组必需的启动因子（IF）瞬时相互作用 与核糖体亚单位和一个专门的起始tRNA，以指导他们的装配在起始密码子 要翻译的mRNA。虽然有证据表明，IFs，核糖体亚基和tRNA经历了 在这个过程中，功能上重要的结构重排，这些重排中很少有 被直接观察和/或表征，对于那些，它一直在非常低的分辨率。 该项目的长期目标是使用强大的试剂和技术组合， 在我们和我们的合作者的实验室中是独一无二的， 启动机制研究。具体来说，我们将使用最先进的单分子荧光 显微镜和低温电子显微镜（cryo-EM），包括开创性的时间分辨cryo-EM 由我们的合作者Joachim Frank博士开发的方法，直接观察和表征动态 开始。这些研究将通过我们开发的新方法来实现， 荧光团进入核糖体的位置是非常可取的，但迄今为止仍然遥不可及。 在目标1中，我们将研究细菌IF 2瞬时结合核糖体的机制。 基于小的30 S核糖体亚基（30 S IC）的起始复合物（IC）;确定30 S IC是否是 携带与正确选择的起始密码子正确碱基配对的准确选择的起始tRNA; 如果是这样，则募集并促进50 S核糖体大亚基与30 S IC的连接。在目标2中，我们 研究细菌IF 3和IF 1确保起始密码子准确性的机制， 在启动时被选中。在目标3中，我们将扩展我们的研究，以探讨真核生物的机制， 翻译启动，把我们的注意力集中在真核生物的具体方面的机制，通过该机制， IF 2的真核同源物，真核IF 5 B（eIF 5 B），在真核起始期间调节亚基连接。

PROJECT SUMMARY The process through which the two-subunit ribosome assembles at the start codon of an mRNA to initiate protein synthesis is one of the most fundamental and highly regulated steps of gene expression. As such, initiation serves as the target of numerous small-molecule antibiotics and cellular pathogens. Moreover, deregulation of initiation is causally linked to viral infections and tumorigenesis in humans. Given all of this, studies of the molecular mechanism of initiation hold great promise for the identification and characterization of mechanistic steps that can serve as targets for the development of next-generation antibiotics and other small- molecule, anti-viral, and anti-cancer drugs that act by modulating translation initiation. Despite their promise for molecular medicine, mechanistic studies of initiation remain incredibly challenging. This is primarily because initiation is an extraordinarily dynamic, multi-step process that proceeds through a large number of short-lived intermediate states that are very difficult to observe and characterize using conventional approaches. During initiation, a set of essential initiation factors (IFs) transiently interact with both ribosomal subunits and a specialized initiator tRNA in order to guide their assembly at the start codon of the mRNA to be translated. Although evidence suggests that the IFs, ribosomal subunits, and tRNA undergo functionally important structural rearrangements during this process, very few of these rearrangements have been directly observed and/or characterized, and for those that have, it has been at very low resolution. The long-term goals of this project are to use powerful combinations of reagents and techniques that are uniquely available in our and our collaborators’ laboratories to overcome the challenges associated with mechanistic studies of initiation. Specifically, we will use state-of-the-art single-molecule fluorescence microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics of initiation. These studies will be enabled by a new approach that we have developed for introducing fluorophores into ribosomes at positions that are highly desirable, but that have thus far remained out of reach. In Aim 1, we will investigate the mechanism through which bacterial IF2 transiently binds to a ribosomal initiation complex (IC) based on the small, 30S, ribosomal subunit (30S IC); determines whether the 30S IC is carrying an accurately selected initiator tRNA that is properly base-paired to a correctly selected start codon; and, if so, recruits and facilitates joining of the large, 50S, ribosomal subunit to the 30S IC. In Aim 2, we will investigate the mechanism through which bacterial IF3 and IF1 ensure the accuracy with which the start codon is selected during initiation. In Aim 3, we will extend our studies to investigate the mechanism of eukaryotic translation initiation, focusing our attention on eukaryotic-specific aspects of the mechanism through which the eukaryotic homolog of IF2, eukaryotic IF5B (eIF5B), regulates subunit joining during eukaryotic initiation.