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: 10266534
• 负责人: Jon Lorsch
• 金额: $ 67.69万
• 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/10266534
• 关键词: 7-methylguanosine; Alanine; Amino Acids; 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; Individual; Initiator Codon; Initiator tRNA; Measures; Messenger RNA; Molecular; Molecular Conformation; Movement; Mutagenesis; Mutation; N-terminal; 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-Binding Proteins; Research; Ribosomes; Role; Saccharomyces cerevisiae; Scaffolding Protein; Scanning; Side; Site; System; Tail; Transfer RNA; Translation Initiation; Yeasts; eIF-4B; eukaryotic initiation factor-5B; genetic approach; genome-wide; inorganic phosphate; mRNA Precursor; molecular mechanics; mutant; reconstitution; recruit; structural biology

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. This year we made significant progress on studies of the N-terminal tail (NTT) or eIF1. This tail appears to play a critical role in responding to recognition of the start codon in the mRNA by the PIC. We used an alanine-scanning mutagenesis approach to determine the effect of changing each amino acid in the tail to alanine. We have identified a variety of phenotypes of these mutants, from lethality to effects on the fidelity of start codon recognition. We are now in the process of studying the effects of key NTT mutations on the function of eIF1 in the in vitro reconstituted yeast translation initiation system in order to help elucidate the mechanistic roles of individual side chains in the NTT. We have also made progress on our in vitro RecSeq system for measuring the efficiency of mRNA recruitment genome wide. This system allows us to isolate the mRNA recruitment steps of translation initiation from later steps in the process and events such as mRNA synthesis or decay, and to actually measure the rates of recruitment of each mRNA simultaneously. Because the system is reconstituted, we can change the concentrations of components or replace them with mutant versions in order to conduct detailed mechanistic analyses.

我们研究小组的目的是阐明真核生物中蛋白质合成的起始阶段的分子机制。我们将酿酒酵母的酵母菌作为模型系统，并采用各种方法 - 从遗传学到生物化学再到结构生物学 - 与NICHD和全球其他几个研究小组的Alan Hinnebusch和Tom Devers Labs合作。 真核翻译引发是基因表达调节的关键控制点。当将引发剂甲基二酮tRNA（met-tRNAI）加载到小（40s）核糖体亚基上时，它就开始了。 Met-tRNAi与40S亚基作为三元络合物（TC）结合了启动因子EIF2的GTP结合形式。 EIF1，EIF1A和EIF3的其他三个因素也与40S亚基结合并促进TC的负载。然后将产生的43S启动络合物（PIC）加载到mRNA的5端，借助EIF3和EIF4组RNA Helicase EIF4A； 5-7-甲基鸟苷帽结合蛋白EIF4E；脚手架蛋白EIF4G；以及40S亚基和RNA结合蛋白EIF4B。 EIF4A和EIF4E都与EIF4G结合并形成EIF4F复合物。一旦装载到mRNA上，就可以认为43S PIC沿mRNA扫描以寻找Aug Start密码子。该过程依赖于ATP，可能需要多种RNA解旋酶，包括Dead-Box蛋白DED1P。识别起点位点始于TRNAI和AUG密码子之间的基础配对。然后，这种基础配对触发了下游事件，这些事件将图片从mRNA上开始持续开始。这些事件包括从其在40S亚基上的结合位点射出EIF1，EIF1A的C末端尾部（CTT）的运动以及从EIF2释放磷酸盐，将其转换为GDP结合状态。此外，引发剂tRNA从没有完全参与核糖体P位点（称为p（out））的位置转移到（p（in）），而整个图片从一个开放的构型转换为有助于扫描到封闭的构型。在此阶段，EIF2GDP与PIC和EIF1A分离，第二个GTPase因子EIF5B，大型核糖体亚基的坐标连接形成80年代的启动复合物。 EIF5B水解GTP，似乎导致复合物的构象重组，然后与EIF1A一起解离。 今年，我们在N末端尾巴（NTT）或EIF1的研究方面取得了重大进展。该尾巴似乎在响应图片中对mRNA中开始密码子的识别方面起着至关重要的作用。我们使用丙氨酸扫描诱变方法来确定改变尾巴中每个氨基酸的作用。我们已经确定了这些突变体的各种表型，从致死性到对起始密码子识别的忠诚度的影响。现在，我们正在研究关键NTT突变对EIF1在体外重构酵母翻译起始系统中的功能的影响，以帮助阐明单个侧链在NTT中的机械作用。 我们还在体外RECSEQ系统上取得了进展，以测量mRNA募集基因组宽的效率。该系统使我们能够将翻译启动的mRNA募集步骤从过程中的后期步骤和mRNA合成或衰减等事件中分离出来，并实际上同时测量每个mRNA的募集速率。由于系统是重组的，因此我们可以更改组件的浓度或用突变版本替换它们以进行详细的机械分析。