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

基本信息

批准号：
8941570
负责人：
Jon Lorsch
金额：
$ 71.63万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/8941570
关键词：
7-methylguanosine Affect Anticodon Base Pairing Binding Binding Sites Biochemistry Biological Models Boxing C-terminal Codon Nucleotides Collaborations Commit Communication Complex Cryoelectron Microscopy Data Elements Event GTP Binding GTPase-Activating Proteins Gene Expression Regulation Genetic Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hydrolysis In Vitro Initiator Codon Initiator tRNA Length Light Messenger RNA Molecular Molecular Conformation Movement Mutation National Institute of Child Health and Human Development Organism Peptide Initiation Factors Phase Phenotype Play Positioning Attribute Process Protein Biosynthesis Proteins RNA Cap-Binding Proteins RNA Helicase RNA, Transfer, Met RNA-Binding Proteins Relative (related person)Research Resolution Role Saccharomyces cerevisiae Scaffolding Protein Scanning Site Staging Structure Suppressor Mutations Tail Transfer RNA Translation Initiation Work Yeasts base conformational conversion eIF-4B eukaryotic initiation factor-5B in vivo inorganic phosphate meetings molecular mechanics response stem structural biology

项目摘要

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. Advances in understanding the mechanism of start codon recognition In collaboration with Alan Hinnebuschs lab, we have probed the functions of conserved identity element bases in the initiator tRNA. Our data indicate that each region of the tRNA plays important roles in start codon recognition. Changing a conserved G:C base pair in the anticodon stem to another base pair decreases the fidelity of start codon recognition (Sui- phenotype), whereas disrupting the base pair increases fidelity (Ssu- phenotype). The latter effect is suppressed by substitutions in a conserved base in the T-loop, indicating long-range communication within the tRNA body. These in vivo effects are mirrored by effects observed in vitro on the stability of TC binding to the 43S PIC, suggesting that the substitutions alter the relative stabilities of the P(OUT) and P(IN) states of the complex. Similarly, changes in the acceptor stem base pair C3:G70 reduce the fidelity of start codon recognition in vivo and perturb TC binding to the PIC in vitro. Overall, our results indicate that, over its entire length, the initiator tRNA structure is finely tuned to set the energetic bars for undergoing the conformational transitions within the PIC that are required for accurate start codon recognition. We also probed the mechanism of action of eIF5, which is the GTPase activating protein for eIF2. In addition to this function, eIF5 has also been shown to play an important role in start codon recognition. In collaboration with Alan Hinnebuschs lab, we showed that the G31R mutation in eIF5, which reduces the fidelity of start codon recognition in vivo by specifically increasing utilization of UUG codons, increases the rate of release of inorganic phosphate from eIF2 in PICs at UUG codons and decreases it at AUG codons. This effect is consistent with our previous work, which indicated that the mutation stabilizes the closed state of the PIC at UUG codons and destabilized it at AUG codons. A suppressor of the G31R mutation, G62S, reverses both of these effects and restores a more normal AUG/UUG initiation ratio in vivo. A second suppressor mutation in eIF5, M18V, functions in a different manner, by diminishing the ability of the factor to activate GTP hydrolysis by eIF2 without affecting the ability of PICs to convert from the open to closed states. Our data support the notion that eIF5 plays multiple, key roles in start codon recognition: activating GTP hydrolysis, controlling phosphate release and modulating the conformational state of the PIC. Finally, in collaboration with Venki Ramakrishnans lab at the MRC in Cambridge, UK, we have determined the structures of intermediate PICs at 4 angstroms resolution using cryo-electron microscopy. These structures shed considerable light on events that take place in the PIC in response to start codon recognition.

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。起始密码子识别机制的研究进展我们与 Alan Hinnebuschs 实验室合作，探讨了起始 tRNA 中保守的同一元件碱基的功能。我们的数据表明 tRNA 的每个区域在起始密码子识别中都发挥着重要作用。将反密码子茎中保守的 G:C 碱基对更改为另一个碱基对会降低起始密码子识别的保真度（Su 表型），而破坏碱基对会增加保真度（Ssu 表型）。后一种效应被 T 环中保守碱基的取代所抑制，表明 tRNA 体内存在长距离通讯。这些体内效应与体外观察到的 TC 与 43S PIC 结合稳定性的效应相一致，表明取代改变了复合物 P(OUT) 和 P(IN) 状态的相对稳定性。同样，受体茎碱基对 C3:G70 的变化会降低体内起始密码子识别的保真度，并扰乱 TC 与体外 PIC 的结合。总体而言，我们的结果表明，在其整个长度上，起始子 tRNA 结构经过精细调整，以设置能量条，以便在 PIC 内进行构象转变，这是准确识别起始密码子所需的。我们还探讨了 eIF5（eIF2 的 GTPase 激活蛋白）的作用机制。除了此功能外，eIF5 还被证明在起始密码子识别中发挥着重要作用。与 Alan Hinnebuschs 实验室合作，我们发现 eIF5 中的 G31R 突变通过专门增加 UUG 密码子的利用率来降低体内起始密码子识别的保真度，增加了 PIC 中 UUG 密码子处的 eIF2 释放无机磷酸盐的速率，并且在 AUG 密码子处减少它。这种效应与我们之前的工作一致，表明突变稳定了 UUG 密码子处 PIC 的闭合状态，并使其在 AUG 密码子处不稳定。 G31R 突变的抑制剂 G62S 可逆转这两种效应并恢复体内更正常的 AUG/UUG 起始比率。 eIF5 中的第二个抑制突变 M18V 以不同的方式发挥作用，即减弱该因子通过 eIF2 激活 GTP 水解的能力，而不影响 PIC 从开放状态转换为封闭状态的能力。我们的数据支持这样的观点，即 eIF5 在起始密码子识别中发挥多种关键作用：激活 GTP 水解、控制磷酸盐释放和调节 PIC 的构象状态。最后，我们与英国剑桥 MRC 的 Venki Ramakrishnans 实验室合作，使用冷冻电子显微镜以 4 埃分辨率确定了中间 PIC 的结构。这些结构为 PIC 响应起始密码子识别而发生的事件提供了相当多的线索。