Alternative mechanisms of different stages in eukaryotic translation

起始是真核生物蛋白质合成中最复杂、最受严格调控的阶段。该过程开始于 在mRNA的起始密码子处形成48 S起始复合物（48 S IC）。第一，43 S预启动 复合物（43 S PIC），包括40 S核糖体亚基、eIF 2·GTP·Met-tRNAMeti三元复合物和 真核生物起始因子eIF 3、eIF 1和eIF 1A在一个步骤中与mRNA的近帽区结合， 由eIFs 4A、4 B和4F介导，其协同展开帽近端区域，允许43 S PIC 协会然后，43 S PIC向下游扫描至起始密码子，在那里它与起始密码子形成48 S IC。 密码子-反密码子相互作用。结构化mRNA的扫描还需要DExH盒 蛋白质DHX 29直接与40 S亚基结合。eIFs 1和1A在确保初始化的精确性方面发挥着关键作用 密码子选择起始密码子识别触发eIF 1解离，eIF 5诱导eIF 2结合的水解 GTP和Pi的释放。60 S亚基的后续连接由翻译的GTIF 5 B促进。 一些病毒mRNA的起始由内部核糖体进入位点（IRES）介导。IRES是高度结构化的 通过非典型相互作用促进40 S亚基的5 '端独立募集的RNA元件 与40 S亚基和/或eIFs连接。翻译起始的失调经常被观察到， 疾病，因此正在成为化疗干预的焦点。虽然所需的因素 已经确定了起始，并确定了它们的主要作用， 机制、监管和替代模式仍然未知。因此，这些细节的表征是 要务我们已经在体外重建了整个翻译周期，这为我们提供了独特的机会， 解决在理解哺乳动物启动和调节的机制方面的关键差距， 使用生物化学和互补生物物理学和细胞生物学方法进行翻译。目标1关注 通过DHX 29促进扫描的机制的表征，eIF 5 B稳定了细胞上的Met-tRNAiMet。 40 S亚基和这两个因素影响起始密码子的选择。在目标2中，我们将重点研究 在CUG密码子和非AUG三联体上用Leu-tRNALeu进行生理学重要起始的机制 在重复相关的非AUG（RAN）翻译过程中，这发生在转录的mRNA中的扩增重复序列上， 导致严重神经退行性疾病的基因目标3致力于阐明 在位于蟋蟀麻痹病毒RNA的5 'UTR的IRES上起始的分子机制，其具有 独特的结构，我们的初步数据表明，它可以使用新的启动机制。目标4关注点 Schlafen 14的细胞功能和作用机制，一种新的结合80 S核糖体的核糖核酸内切酶 并切割rRNA和核糖体结合的mRNA。因此，它涉及翻译控制，并可能影响 这是一个以前没有描述过的过程。

Initiation is the most complex, tightly regulated stage of eukaryotic protein synthesis. The process begins with formation of the 48S initiation complex (48S IC) at the initiation codon of mRNA. First, the 43S preinitiation complex (43S PIC) comprising the 40S ribosomal subunit, the eIF2•GTP•Met-tRNAMeti ternary complex and eukaryotic initiation factors eIF3, eIF1 and eIF1A binds to the cap-proximal region of mRNA in a step that is mediated by eIFs 4A, 4B and 4F, which cooperatively unwind the cap-proximal region, allowing for 43S PIC association. The 43S PIC then scans downstream to the initiation codon where it forms the 48S IC with the established codon-anticodon interaction. Scanning on structured mRNAs additionally requires the DExH-box protein DHX29 that binds directly to 40S subunits. eIFs 1 and 1A play key roles in ensuring the fidelity of initiation codon selection. Initiation codon recognition triggers dissociation of eIF1, eIF5-induced hydrolysis of eIF2-bound GTP and release of Pi. Subsequent joining of a 60S subunit is promoted by the translational GTPase eIF5B. Initiation on some viral mRNAs is mediated by an internal ribosome entry site (IRES). IRESs are highly structured RNA elements that promote 5’-end independent recruitment of the 40S subunit via non-canonical interactions with the 40S subunits and/or eIFs. Dysregulation of translation initiation is frequently observed in devastating diseases and is therefore becoming a focus for chemo-therapeutic intervention. Although the factors required for initiation have been identified, and their principal roles determined, important details concerning its molecular mechanism, regulation and alternative modes remain unknown. Characterization of these details is therefore a priority. We have reconstituted the entire translation cycle in vitro, which gives us the unique opportunity to address critical gaps in understanding of the mechanisms of mammalian initiation and the regulation of translation using biochemical and complementary biophysical and cell biology approaches. Aim 1 will concern characterization of the mechanisms by which DHX29 promotes scanning, eIF5B stabilizes Met-tRNAiMet on the 40S subunit and both factors influence initiation codon selection. In Aim 2, we will focus on investigating the mechanisms of physiologically important initiation with Leu-tRNALeu at CUG codons, and on non-AUG triplets during repeat-associated non-AUG (RAN) translation, which occurs on expansion repeats in mRNAs transcribed from genes that are responsible for severe neurodegenerative diseases. Aim 3 is devoted to elucidation of the molecular mechanism of initiation on the IRES located in the 5'UTR of Cricket paralysis virus RNA, which has a unique structure and that our preliminary data suggest can use novel mechanisms for initiation. Aim 4 concerns the cellular function and mechanism of action of Schlafen14, a novel endoribonuclease that binds 80S ribosomes and cleaves rRNA and ribosome-bound mRNAs. It is thus implicated in translational control, and likely influences this process in a previously undescribed manner.