Alternative mechanisms of different stages in eukaryotic translation

起始阶段是真核生物蛋白质合成过程中最复杂、调控最严格的阶段。这一过程开始于 在mRNA起始密码子上形成48S起始复合体(48S IC)。第一，43s预印心 包含40S核糖体亚基的复合体(43S PIC)、eIF2·GTP·Met-tRNAMeti三元复合体和 真核启动因子eIF3、eIF1和eIF1a通过一个步骤与mRNA的帽-近端区域结合，即 由EIFS4A、4B和4F介导，它们协同解开帽状近端区域，允许43S PIC 协会。然后，43S PIC向下游扫描到起始密码子，在那里它与48S IC形成 已建立密码子-反密码子相互作用。扫描结构化的mRNA还需要DExH-box 直接与40S亚基结合的蛋白质DHX29。EIFS1和1A在确保启动的保真度方面发挥了关键作用 密码子选择。起始密码子识别触发eIF1的解离，eIF5诱导eIF2结合的水解 GTP和PI的释放。随后60S亚基的加入由翻译的GTP酶eIF5B促进。 一些病毒mRNAs的起始是由内部核糖体进入位点(IRES)介导的。IRES是高度结构化的 通过非正则相互作用促进40S亚基5‘端独立募集的RNA元件 具有40s亚基和/或EIF。翻译启动的失调在《毁灭性》中经常被观察到 因此，它正成为化疗干预的重点。尽管满足以下要求的因素 有关其分子的重要细节已被确定，并确定了它们的主要作用 机制、监管和替代模式仍不清楚。因此，对这些细节的描述是一个 优先考虑。我们已经在体外重建了整个翻译周期，这给了我们独特的机会 解决在理解哺乳动物的启动和调控机制方面的关键差距 使用生化和互补的生物物理和细胞生物学方法进行翻译。目标1将关注 DHX29促进扫描的机制表征，eIF5B稳定Met-tRNAiMet在 40S亚基和这两个因素都影响起始密码子的选择。在目标2中，我们将重点调查 CUG密码子和非AUG三联体上Leu-tRNALeu的重要生理启动机制 在重复相关的非8月(RAN)翻译期间，这发生在转录的mRNA中的扩展重复上 来自导致严重神经退行性疾病的基因。目标3致力于阐明 蟋蟀麻痹病毒RNA5‘端非编码区IRES启动的分子机制 独特的结构，我们的初步数据表明，可以使用新的机制来启动。目标4关注点 结合80S核糖体的新型内切核酸酶Schlafen14的细胞功能和作用机制 并切割rRNA和核糖体结合的mRNAs。因此，它牵涉到翻译控制，并可能影响 这一过程以一种以前未曾描述过的方式。

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.