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: 10685193
• 负责人: Jon Lorsch
• 金额: $ 62.73万
• 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/10685193
• 关键词: 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; Initiator Codon; Initiator tRNA; Messenger RNA; Methodology; 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; Reproducibility; Research; Ribosomes; Saccharomyces cerevisiae; Scaffolding Protein; Scanning; Site; Tail; Transfer RNA; Translation Initiation; Work; Yeasts; eIF-4B; eukaryotic initiation factor-5B; genetic approach; inorganic phosphate; molecular mechanics; 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. In 2021, we continued to use our transcriptome-wide approach to monitoring mRNA recruitment to 43S PICs, RecSeq, to study the translation initiation process. We honed the methodology to make it more reproducible. We have been using it to study the effects of 40S subunit concentration on the rates of mRNA recruitment in order to understand how "weak" versus "strong" mRNAs respond to changes in ribosome abundance. We have also been finishing work on using RecSeq to study the effects of the RNA helicase Ded1 on the recruitment of mRNAs transcriptome wide. We hope to finish the latter studies soon.

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。 在2021年，我们继续使用全转录组的方法来监测43S图片RecSeq的mRNA募集，以研究翻译起始过程。我们磨练了使其更可重现的方法。我们一直在使用它来研究40S亚基浓度对mRNA募集速率的影响，以了解“弱”与“强” mRNA如何响应核糖体丰度的变化。我们还一直在完成使用RECSEQ来研究RNA解旋酶DED1对mRNA转录组宽的募集的影响。我们希望尽快完成后一种研究。