Alternative mechanisms of different stages in eukaryotic translation

真核翻译不同阶段的替代机制

基本信息

批准号：
10161790
负责人：
CHRISTOPHER Ulrich Tristram HELLEN
金额：
$ 32.3万
依托单位：
SUNY DOWNSTATE MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10161790
关键词：
5&apos Untranslated Regions Address Anticodon Binding Biochemical Biological Biophysics Cell physiology Cellular biology Cleaved cell Codon Nucleotides Complex Cryoelectron Microscopy Data Development Dipeptides Disease Dissociation Drug Targeting Electron Transport Complex III Elements Endoribonucleases Ensure Eukaryotic Initiation Factors GTP Binding Gene Expression Gene Expression Regulation Genes Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Homeostasis Hydrolysis In Vitro Individual Infection Inherited Initiator Codon Insecta Interferons Internal Ribosome Entry Site Intervention Leucine-Specific tRNA Malignant Neoplasms Mammalian Cell Mammals Mediating Messenger RNA Missense Mutation Molecular Molecular Analysis N-terminal Natural Immunity Neurodegenerative Disorders Open Reading Frames Pathogenesis Pathologic Pathway interactions Physiological Play Process Protein Biosynthesis Proteins RNA Regulation Ribosomal RNA Ribosomes Role Scanning Site Stress Structural Protein Structure Structure-Activity Relationship System Thrombocytopenia Transfer RNA Translation Initiation Translations Triplet Multiple Birth Untranslated Regions Viral biophysical techniques clinically relevant cricket paralysis virus eukaryotic initiation factor-5B improved influenza infection insight novel reconstitution recruit response tool translatome viral RNA

项目摘要

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.

起始是真核蛋白质合成中最复杂、受严格调控的阶段。在mRNA起始密码子处形成48S起始复合物(48S IC) 首先，43S预起始。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 然后 43S PIC 向下游扫描到起始密码子，并在其中与 48S IC 形成关联。已建立的密码子-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 在确保起始保真度方面发挥着关键作用。起始密码子识别触发 eIF1 解离，eIF5 诱导 eIF2 结合的水解。 GTP 和 Pi 的随后加入由翻译 GTPase eIF5B 促进。一些病毒 mRNA 的启动是由高度结构化的内部核糖体进入位点 (IRES) 介导的。通过非规范相互作用促进 40S 亚基 5' 端独立招募的 RNA 元件 40S 亚基和/或 eIF 的翻译起始失调经常在破坏性中观察到。疾病，因此成为化疗干预的重点。启动已被确定，其主要作用已确定，有关其分子的重要细节因此，这些细节的机制、监管和替代模式仍然未知。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对哺乳动物起始机制和调节的理解方面的关键差距目标 1 将涉及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。 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，我们将重点研究 40S 亚基。在 CUG 密码子和非 AUG 三联体上使用 Leu-tRNALeu 的生理学重要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 的扩展重复上目标 3 致力于阐明导致严重神经退行性疾病的基因。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机制来启动目标 4。 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，并可能产生影响。该过程以先前未描述的方式进行。