THE MECHANISMS OF EUKARYOTIC TRANSLATION TERMINATION AND RIBOSOMAL RECYCLING

真核翻译终止和核糖体回收的机制

• 批准号: 8538426
• 负责人: TATYANA V PESTOVA
• 金额: $ 36.19万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538426
• 关键词: Antibiotics; Binding; Biochemical; Biological; Boxing; Cells; Codon Nucleotides; Complex; Couples; Cystic Fibrosis; DNA Sequence Rearrangement; Data; Defect; Development; Disease; Dissociation; Elements; Enzymes; Family; Funding; GTP Binding; Gene Expression; Genetic Transcription; Guanosine Triphosphate; Hereditary Disease; Human; Hydrolysis; In Vitro; Individual; Inherited; Investigation; Length; Mediating; Messenger RNA; Modeling; Muscular Dystrophies; Mutation; Nonsense Codon; Nonsense-Mediated Decay; Open Reading Frames; Peptides; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Process; Protein Biosynthesis; Protein Family; Proteins; RNA Helicase; RNA Splicing; Recycling; Regulation; Relative (related person); Ribosomes; Role; Site; Staging; Stress; Structure; System; Terminator Codon; Trans-Activators; Transfer RNA; Translation Process; Translations; Virus; base; cis acting element; clinically relevant; cofactor; derepression; in vivo; mRNA Decay; mRNA Surveillance; mRNA Transcript Degradation; member; nuclease; peptidyl-tRNA; programs; public health relevance; reconstitution; release factor; ribosome releasing factor; thrombocytosis; transcription factor

DESCRIPTION (provided by applicant): Eukaryotic termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition and peptidyl-tRNA hydrolysis by eRF1. Recycling of post-termination complexes (post-TCs) is promoted by the concerted action of ABCE1, an essential member of ATP-binding cassette family of proteins, and eRF1, which remains associated with post- TCs after peptide release. eRF1 thus participates in two successive stages of protein synthesis, termination and ribosome recycling. In some cases post-TCs do not undergo complete recycling, and termination is instead followed by reinitiation on the same mRNA, usually downstream of the stop codon. This mostly occurs after translation of short open reading frames (ORFs), whereas reinitiation after long ORFs is rare and appears to be limited to several virus families. Termination/recycling are also associated with two other important processes, mRNA surveillance by No-Go and nonsense-mediated decay (NGD and NMD, respectively). NGD targets mRNAs on which elongation complexes (ECs) are stalled by e.g. stable secondary structure. It is initiated by endonucleolytic cleavage in the vicinity of stalled ribosomes, which s stimulated by eRF1 and eRF3 paralogues Pelota and Hbs1 that together with ABCE1 can then dissociate ECs without prior peptide release. NMD targets aberrant mRNAs containing premature termination codons (PTCs), which are introduced by aberrant splicing, mutation or transcription errors. Recognition and targeting for degradation of PTC-containing mRNAs is mediated by conserved effectors, including UPF1 (a DEAD-box RNA helicase), UPF2, UPF3B and the UPF1 kinase SMG1. UPF1 interacts with eRF1, eRF3, UPF2, UPF3B and SMG1, and plays a key role in the process. It was suggested that ribosomes arrested at PTCs are recognized by a SURF (SMG1, UPF1, eRF1/3) surveillance complex, and that recruitment of UPF1 also impairs termination. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosoma recycling) from individual purified translational components. In Aim 1, we propose to investigate the structure of ribosome recycling and termination complexes corresponding to different stages in the process, and to characterize how termination is regulated by cis-acting mRNA elements and trans-acting factors. In Aim 2 we will recapitulate different modes of reinitiation using our i vitro reconstituted system to determine their factor requirements and mechanisms. In Aim 3 we will define the requirements for ABCE1/Pelota-mediated dissociation of stalled ribosomal elongation complexes, and will undertake an attempt to localize the site of endonucleolytic cleavage in NGD and to identify the responsible nuclease. Aim 4 will be devoted to investigation of the influence of UPF1, depending on its phosphorylation status and the presence of UPF2 and UPF3 cofactors, on individual steps in the translation process.

描述（由申请人提供）：真核终止是由两个释放因子 eRF1 和 eRF3 之间复杂的功能相互作用造成的，其中 eRF3 的 GTP 水解与 eRF1 的密码子识别和肽基-tRNA 水解相结合。 ABCE1（蛋白质 ATP 结合盒家族的重要成员）和 eRF1（在肽释放后仍与后 TC 相关）的协同作用促进了终止后复合物（后 TC）的回收。因此，eRF1 参与蛋白质合成、终止和核糖体回收的两个连续阶段。在某些情况下，后 TC 不会完全回收，终止后会在相同的 mRNA 上重新启动，通常位于终止密码子的下游。这主要发生在短开放阅读框 (ORF) 翻译后，而长 ORF 后重新启动的情况很少见，并且似乎仅限于几个病毒家族。终止/回收还与另外两个重要过程相关，即 No-Go 的 mRNA 监视和无义介导的衰变（分别为 NGD 和 NMD）。 NGD 靶向 mRNA，其上的延伸复合物 (EC) 被例如稳定的二级结构。它是由停滞核糖体附近的核酸内切裂解引发的，该裂解受到 eRF1 和 eRF3 旁系同源物 Pelota 和 Hbs1 的刺激，然后与 ABCE1 一起可以解离 EC，而无需事先释放肽。 NMD 的目标是含有过早终止密码子 (PTC) 的异常 mRNA，这些密码子是由异常剪接、突变或转录错误引入的。含 PTC mRNA 降解的识别和靶向是由保守效应子介导的，包括 UPF1（DEAD-box RNA 解旋酶）、UPF2、UPF3B 和 UPF1 激酶 SMG1。 UPF1与eRF1、eRF3、UPF2、UPF3B和SMG1相互作用，并在此过程中发挥关键作用。有人建议，在 PTC 处捕获的核糖体可被 SURF（SMG1、UPF1、eRF1/3）监视复合体识别，并且 UPF1 的募集也会损害终止。拟议的研究将基于从单个纯化的翻译成分中体外重建蛋白质合成的所有阶段（起始、延伸、终止和核糖体回收）的方法。在目标 1 中，我们建议研究对应于该过程中不同阶段的核糖体回收和终止复合物的结构，并表征顺式作用 mRNA 元件和反式作用因子如何调节终止。在目标 2 中，我们将使用我们的体外重构系统来概括不同的重新启动模式，以确定其因子要求和机制。在目标 3 中，我们将定义 ABCE1/Pelota 介导的停滞核糖体延伸复合物解离的要求，并将尝试定位 NGD 中核酸内切切割的位点并确定负责的核酸酶。目标 4 将致力于研究 UPF1 对翻译过程中各个步骤的影响，具体取决于其磷酸化状态以及 UPF2 和 UPF3 辅因子的存在。