Elucidating the Molecular Mechanics of Eukaryotic Translation Initiation and Its Control

阐明真核翻译起始及其控制的分子机制

• 批准号: 10471708
• 负责人: Jon Lorsch
• 金额: $ 57.77万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471708
• 关键词: 5' Untranslated Regions; 7-methylguanosine; Anticodon; Base Pairing; Binding; Binding Sites; Biochemistry; Biological Models; C-terminal; Codon Nucleotides; Collaborations; Complex; Event; GTP Binding; Gene Expression Regulation; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; In Vitro; Initiator Codon; Initiator tRNA; Kinetics; Length; Measurement; Messenger RNA; Molecular; Molecular Conformation; Monitor; Movement; National Institute of Child Health and Human Development; Organism; Peptide Initiation Factors; Phase; Positioning Attribute; Process; Protein Biosynthesis; Proteins; RNA Cap-Binding Proteins; RNA Helicase; RNA-Binding Proteins; Research; Ribosomes; Role; Saccharomyces cerevisiae; Scaffolding Protein; Scanning; Site; Structure; Tail; Techniques; Transfer RNA; Translation Initiation; Yeasts; eIF-4B; eukaryotic initiation factor-5B; genetic approach; inorganic phosphate; molecular mechanics; pandemic disease; recruit; structural biology; transcriptome

The goal of our research group is to elucidate the molecular mechanisms underlying the initiation phase of protein synthesis in eukaryotic organisms. We use the yeast saccharomyces cerevisiae as a model system and employ a range of approaches - from genetics to biochemistry to structural biology - in collaboration with Alan Hinnebusch and Tom Devers labs at NICHD and several other research groups around the world. Eukaryotic translation initiation is a key control point in the regulation of gene expression. It begins when an initiator methionyl tRNA (Met-tRNAi) is loaded onto the small (40S) ribosomal subunit. Met-tRNAi binds to the 40S subunit as a ternary complex (TC) with the GTP-bound form of the initiation factor eIF2. Three other factors eIF1, eIF1A and eIF3 also bind to the 40S subunit and promote the loading of the TC. The resulting 43S pre-initiation complex (PIC) is then loaded onto the 5-end of an mRNA with the aid of eIF3 and the eIF4 group of factors the RNA helicase eIF4A; the 5-7-methylguanosine cap-binding protein eIF4E; the scaffolding protein eIF4G; and the 40S subunit- and RNA-binding protein eIF4B. Both eIF4A and eIF4E bind to eIF4G and form the eIF4F complex. Once loaded onto the mRNA, the 43S PIC is thought to scan along the mRNA in search of an AUG start codon. This process is ATP-dependent and likely requires multiple RNA helicases, including the DEAD-box protein Ded1p. Recognition of the start site begins with base pairing between the anticodon of tRNAi and the AUG codon. This base pairing then triggers downstream events that commit the PIC to continuing initiation from that point on the mRNA. These events include ejection of eIF1 from its binding site on the 40S subunit, movement of the C-terminal tail (CTT) of eIF1A, and release of phosphate from eIF2, which converts it to its GDP-bound state. In addition, the initiator tRNA moves from a position that is not fully engaged in the ribosomal P site (termed P(OUT)) to one that is (P(IN)) and the PIC as a whole converts from an open conformation that is conducive for scanning to a closed one that is not. At this stage eIF2GDP dissociates from the PIC and eIF1A and a second GTPase factor, eIF5B, coordinate joining of the large ribosomal subunit to form the 80S initiation complex. eIF5B hydrolyzes GTP, which appears to result in a conformational reorganization of the complex, and then dissociates along with eIF1A. Despite restrictions put in place due to the pandemic, we were able to make progress this year. We adapted our transcriptome-wide technique RecSeq, which allows us to monitor mRNA recruitment to the 43S PIC in vitro, to perform studies of the kinetics of mRNA recruitment. This approach has allowed us to group mRNAs according to their rates of recruitment, with most binding rapidly to the PIC but a subset binding more slowly. The rate of recruitment correlates negatively with length and the amount of structure in the 5'-untranslated regions of the mRNAs. We are using RecSeq kinetic measurements to probe the roles of factors involved in mRNA recruitment including eIF4A, eIF4B and Ded1.

我们研究小组的目标是阐明真核生物蛋白质合成起始阶段的分子机制。我们使用酿酒酵母作为模型系统，并与 NICHD 的 Alan Hinnebusch 和 Tom Devers 实验室以及世界各地的其他几个研究小组合作，采用了从遗传学到生物化学到结构生物学的一系列方法。 真核翻译起始是基因表达调控的关键控制点。当起始子甲硫氨酰 tRNA (Met-tRNAi) 被加载到小 (40S) 核糖体亚基上时，它就开始了。 Met-tRNAi 以三元复合物 (TC) 的形式与 40S 亚基与起始因子 eIF2 的 GTP 结合形式结合。其他三个因子 eIF1、eIF1A 和 eIF3 也与 40S 亚基结合并促进 TC 的加载。然后，借助 eIF3 和 eIF4 因子（RNA 解旋酶 eIF4A），将所得 43S 前起始复合物 (PIC) 加载到 mRNA 的 5 端； 5-7-甲基鸟苷帽结合蛋白 eIF4E；支架蛋白 eIF4G；以及 40S 亚基和 RNA 结合蛋白 eIF4B。 eIF4A 和 eIF4E 均与 eIF4G 结合并形成 eIF4F 复合物。一旦加载到 mRNA 上，43S PIC 就会沿着 mRNA 扫描以寻找 AUG 起始密码子。该过程依赖于 ATP，并且可能需要多个 RNA 解旋酶，包括 DEAD-box 蛋白 Ded1p。起始位点的识别从 tRNAi 的反密码子和 AUG 密码子之间的碱基配对开始。然后，这种碱基配对会触发下游事件，使 PIC 从 mRNA 上的该点继续启动。这些事件包括 eIF1 从 40S 亚基上的结合位点弹出、eIF1A C 末端尾部 (CTT) 的运动以及 eIF2 释放磷酸盐，从而将其转化为 GDP 结合状态。此外，起始子 tRNA 从未完全参与核糖体 P 位点的位置（称为 P(OUT)）移动到 (P(IN))，并且 PIC 作为一个整体从开放构象转变为有利于扫描到一个不封闭的。在此阶段，eIF2GDP 从 PIC 和 eIF1A 解离，第二个 GTP 酶因子 eIF5B 协调大核糖体亚基的连接，形成 80S 起始复合物。 eIF5B 水解 GTP，这似乎会导致复合物的构象重组，然后与 eIF1A 一起解离。 尽管由于大流行而实施了限制，但我们今年还是取得了进展。我们采用了全转录组技术 RecSeq，该技术使我们能够在体外监测 43S PIC 的 mRNA 募集，以研究 mRNA 募集的动力学。这种方法使我们能够根据 mRNA 的招募速率对 mRNA 进行分组，其中大多数 mRNA 快速与 PIC 结合，但有一部分 mRNA 结合速度较慢。招募率与 mRNA 5'-非翻译区的长度和结构量呈负相关。我们正在使用 RecSeq 动力学测量来探讨参与 mRNA 招募的因素（包括 eIF4A、eIF4B 和 Ded1）的作用。