Elucidating the Molecular Mechanics of Eukaryotic Translation Initiation and Its Control

我们研究小组的目标是阐明真核生物中蛋白质合成起始阶段的分子机制。我们使用酵母酿酒酵母作为模型系统，并采用一系列方法-从遗传学到生物化学到结构生物学-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。 真核翻译起始是基因表达调控的关键控制点。它开始时，引发剂甲硫氨酰tRNA（Met-tRNAi）加载到小（40 S）核糖体亚基。Met-tRNAi与起始因子eIF 2的GTP结合形式作为三元复合物（TC）结合至40 S亚基。另外三种因子eIF 1、eIF 1A和eIF 3也与40 S亚基结合，并促进TC的负载。然后，在eIF 3和eIF 4组因子的帮助下，将所得的43 S前起始复合物（PIC）加载到mRNA的5-末端上，所述eIF 4组因子为RNA解旋酶eIF 4A、5-7-甲基鸟苷帽结合蛋白eIF 4 E、支架蛋白eIF 4G以及40 S亚基和RNA结合蛋白eIF 4 B。eIF 4A和eIF 4 E均与eIF 4G结合并形成eIF 4F复合物。一旦加载到mRNA上，43 S PIC被认为沿着mRNA扫描以寻找AUG起始密码子。这个过程是ATP依赖性的，可能需要多种RNA解旋酶，包括DEAD盒蛋白Ded 1 p。起始位点的识别开始于tRNAi的反密码子和AUG密码子之间的碱基配对。然后，这种碱基配对触发下游事件，使PIC从mRNA上的该点继续起始。这些事件包括eIF 1从其40 S亚基上的结合位点排出，eIF 1A的C-末端尾（CTT）的移动，以及从eIF 2释放磷酸盐，将其转化为GDP结合状态。此外，起始tRNA从一个不完全接合在核糖体P位点的位置（称为P（OUT））移动到一个完全接合在核糖体P位点的位置（称为P（IN）），而PIC作为一个整体从一个有利于扫描的开放构象转变为一个不利于扫描的封闭构象。在这个阶段，eIF 2GDP从PIC和eIF 1A解离，第二个GT3因子eIF 5 B协调连接核糖体大亚基形成80 S起始复合物。eIF 5 B水解GTP，这似乎导致复合物的构象重组，然后沿着eIF 1 A解离。 于二零二一年，我们继续使用全转录组方法监测mRNA向43 S PIC的募集，即RecSeq，以研究翻译起始过程。我们改进了方法，使其更具可重复性。我们一直使用它来研究40 S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。我们还完成了使用RecSeq研究RNA解旋酶Ded 1对mRNA转录组募集的影响的工作。我们希望很快完成后一项研究。

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.