The mechanism and regulation of mRNA recruitment during eukaryotic translation initiation

项目总结 核糖体将信使RNA(MRNAs)翻译成蛋白质的效率是 基因表达的基本决定因素。这种效率通常是在信使核糖核酸的募集过程中决定的。 核糖体翻译的步骤。因此，这一步是基因表达的关键控制点。在……里面 在真核生物中，mRNA的募集是一个复杂的、多步骤的、高度调控的过程，依赖于 ~13个真核起始因子(EIF)的活性。EIF活性和mRNA募集的失调 与肿瘤发生、肿瘤生长、耐药性和转移有因果关系的人越来越多 癌症。因此，几个EIF及其在mRNA招募中的作用正在成为非常有吸引力的 抗癌药物靶点，现有的FDA批准的EIF靶向化合物已经 成功地被重新用于抗癌治疗。为了扩大和充分开发这一治疗潜力， 然而，有必要了解eIF功能和mRNA募集背后的分子事件。 在这里，我们将使用高纯度、荧光团标记的酿酒酵母进行体外翻译 我们开发的系统，包括全长、特定位置标记的eIF4G和完全重组的 定点标记的eIF3，这是一种很难产生的试剂。有了这些试剂，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)， 包括我们的合作者约阿希姆·弗兰克博士开发的开创性、时间分辨的冷冻-EM方法，以 直接观察和表征真核细胞翻译起始过程中的mRNA募集动态。 在目标1中，我们将研究多组分eIF4F复合体激活的机制 用于装载到核糖体43S预起始复合体(PIC)上的不同类别的mRNAs，以及如何改变为 EIF4F复合体的组成可以改变激活哪些类别的mRNAs。我们假设 激活的信使核糖核酸复合体的结构动力学对信使核糖核酸的负载至关重要，并将如何量化这些 动态有助于信使核糖核酸的选择。在目标2中，我们将研究多组分eIF3是如何复合的 与不同类别的mRNAs和/或43S PIC相互作用，以促进mRNAs的激活和/或加载 到43S PIC上，以及eIF3的生物活性亚复合体如何调节这些活性。一个大的- 43S PIC结合的eIF3的尺度结构重排被认为控制其mRNA的负载活性，我们将 因此，请描述这种重排如何促进不同类型的mRNAs上48S PIC的形成 上课。在目标3中，我们将研究eIF1A和eIF5B介导mrna起始密码子的机制。 48s PIC内的识别。EIF1a的起始密码子识别最近与大规模的 EIF5B和启动子转移RNA(Met-tRNAi)在48S PIC中的重排 准备将大亚基连接到48S PIC上，形成具有延伸性的80S IC。我们会 描述这些48S PIC的动力学特征，并确定它们在起始密码子识别和亚基连接中的作用。

PROJECT SUMMARY The efficiency with which messenger RNAs (mRNAs) are translated into proteins by the ribosome is a fundamental determinant of gene expression. This efficiency is often determined during the mRNA recruitment step of translation by the ribosome. Consequently, this step is a crucial point of control for gene expression. In eukaryotes, mRNA recruitment is an elaborate, multi-step, and highly regulated process that depends upon the activities of ~13 eukaryotic initiation factors (eIFs). Dysregulation of eIF activity and mRNA recruitment has been causally linked to tumorigenesis, tumor growth, drug resistance, and metastasis in an increasing list of human cancers. Consequently, several eIFs and their roles in mRNA recruitment are emerging as very attractive anticancer drug targets, with an existing, FDA-approved, eIF-targeting compound already having been successfully repurposed as an anticancer therapy. In order to expand and fully exploit this therapeutic potential, however, it is necessary to understand the molecular events that underlie eIF function and mRNA recruitment. Here, we will use a highly purified, fluorophore-labeled, Saccharomyces cerevisiae in vitro translation system that we developed and that includes a full-length, site-specifically labeled eIF4G and a fully reconstituted, site-specifically labeled eIF3, reagents that have been difficult to generate. With these reagents in hand, 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 mRNA recruitment during eukaryotic translation initiation. In Aim 1, we will investigate the mechanism through which the multi-component eIF4F complex activates different classes of mRNAs for loading onto ribosomal 43S pre-initiation complexes (PICs), and how changes to the composition of the eIF4F complex can alter which classes of mRNAs are activated. We hypothesize that the structural dynamics of the activated mRNA complex are critical for mRNA loading and will quantify how these dynamics contribute to mRNA selection. In Aim 2, we will investigate how the multi-component eIF3 complex interacts with different classes of mRNAs and/or the 43S PIC in order to facilitate mRNA activation and/or loading onto a 43S PIC, as well as how biologically active subcomplexes of eIF3 can modulate these activities. A large- scale structural rearrangement of 43S PIC-bound eIF3 is thought to control its mRNA loading activity and we will therefore characterize how this rearrangement facilitates formation of the 48S PIC on mRNAs of different classes. In Aim 3, we will investigate the mechanism through which eIF1A and eIF5B mediate mRNA start-codon recognition within a 48S PIC. Start-codon recognition by eIF1A has recently been associated with a large-scale rearrangement of the 48S PIC in which eIF5B and initiator transfer RNA (Met-tRNAi) are repositioned in preparation for joining of the large subunit to the 48S PIC to form the elongation-competent 80S IC. We will characterize these 48S PIC dynamics and determine their role in start-codon recognition and subunit joining.