The Structural Dynamics of Translation Initiation

项目总结 双亚单位核糖体在信使核糖核酸的起始密码子上组装成 启动蛋白质合成是基因表达最基本、最受调控的步骤之一。AS 这样的启动是许多小分子抗生素和细胞病原体的靶标。此外， 解除对启动的管制与人类的病毒感染和肿瘤发生有因果关系。考虑到这一切， 研究引发的分子机制对鉴定和表征具有重要意义。 可以作为下一代抗生素和其他小型抗生素开发目标的机械性步骤 通过调节翻译启动起作用的分子、抗病毒和抗癌药物。 尽管他们对分子医学充满希望，但启动的机制研究仍然令人难以置信 很有挑战性。这主要是因为启动是一个非常动态的、多步骤的过程， 通过大量很难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，一组基本启动因子(IF)短暂地相互作用 具有核糖体亚基和专门的启动子tRNA，以引导它们在起始密码子上组装 要翻译的信使核糖核酸。尽管有证据表明IF、核糖体亚基和tRNA经历了 在这一过程中，这些结构调整中很少有 被直接观察和/或表征的，而对于那些已经被观察和/或表征的，其分辨率一直很低。 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独一无二地提供，以克服与 启蒙的机理研究。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、时间分辨的低温电子显微镜 我们的合作者约阿希姆·弗兰克博士开发了一种直接观察和表征动力学的方法 关于入会的。这些研究将通过我们开发的一种新方法来实现 荧光团进入核糖体的位置是非常可取的，但到目前为止还达不到。 在目标1中，我们将研究细菌if2与核糖体瞬时结合的机制。 以小的30S核糖体亚基为基础的起始复合体(IC)；决定30S IC是否 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启动子tRNA； 如果是这样的话，招募并促进50s的大核糖体亚基与30s IC的连接。在目标2中，我们将 探讨细菌IF3和IF1确保起始密码子准确性的机制 在启动过程中被选中。在目标3中，我们将扩展我们的研究，以探索真核生物的机制。 翻译启动，将我们的注意力集中在真核生物特有的机制方面，通过这种机制 IF2的真核同源物，真核IF5B(EIF5B)，在真核启动过程中调节亚基的连接。

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.