Alternative mechanisms of different stages in eukaryotic translation

起始是真核蛋白质合成中最复杂、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48S起始复合物(48S IC)。一、43S预启动 复合物 (43S PIC) 包含 40S 核糖体亚基、eIF2•GTP•Met-tRNAMeti 三元复合物和 真核起始因子 eIF3、eIF1 和 eIF1A 与 mRNA 的近端区结合的步骤是 由 eIF 4A、4B 和 4F 介导，它们协同解开帽近端区域，从而实现 43S PIC 协会。然后 43S PIC 向下游扫描至起始密码子，并在其中形成 48S IC 建立密码子-反密码子相互作用。扫描结构化 mRNA 还需要 DExH-box 直接与 40S 亚基结合的蛋白质 DHX29。 eIF 1 和 1A 在确保起始保真度方面发挥着关键作用 密码子选择。起始密码子识别触发 eIF1 解离，eIF5 诱导 eIF2 结合水解 GTP 和 Pi 的发布。随后 60S 亚基的加入由翻译 GTP 酶 eIF5B 促进。 一些病毒 mRNA 的启动是由内部核糖体进入位点 (IRES) 介导的。 IRES 是高度结构化的 通过非规范相互作用促进 40S 亚基 5' 端独立募集的 RNA 元件 与 40S 亚基和/或 eIF。翻译起始的失调经常在毁灭性的灾难中观察到。 疾病，因此成为化疗干预的焦点。虽然需要的因素 已经确定了起始作用，并确定了它们的主要作用，有关其分子的重要细节 机制、监管和替代模式仍然未知。因此，这些细节的表征是 优先。我们在体外重建了整个翻译周期，这给了我们独特的机会 解决对哺乳动物起始机制和调节的理解方面的关键差距 使用生物化学和互补的生物物理和细胞生物学方法进行翻译。目标1将涉及 DHX29 促进扫描的机制特征，eIF5B 稳定 Met-tRNAiMet 40S 亚基和这两个因素都会影响起始密码子的选择。在目标 2 中，我们将重点调查 在 CUG 密码子和非 AUG 三联体上用 Leu-tRNALeu 进行生理上重要的起始机制 在重复相关的非 AUG (RAN) 翻译过程中，发生在转录的 mRNA 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 位于蟋蟀麻痹病毒RNA 5'UTR的IRES启动的分子机制，它具有 独特的结构，我们的初步数据表明可以使用新颖的引发机制。目标 4 关注点 Schlafen14（一种结合 80S 核糖体的新型核糖核酸内切酶）的细胞功能和作用机制 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 该过程以先前未描述的方式进行。

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.