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: 8727581
• 负责人: TATYANA V PESTOVA
• 金额: $ 37.5万
• 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/8727581
• 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）：真核生物终止是由两种释放因子eRF 1和eRF 3之间的复杂功能相互作用引起的，其中eRF 3的GTP水解耦合了密码子识别和eRF 1的肽基-tRNA水解。终止后复合物（post-TC）的再循环通过ABCE 1（ATP结合盒蛋白家族的重要成员）和eRF 1（其在肽释放后保持与post-TC结合）的协同作用而促进。因此，eRF 1参与蛋白质合成的两个连续阶段，终止和核糖体再循环。在某些情况下，后TC不经历完全的再循环，而是在终止后在相同的mRNA上重新起始，通常在终止密码子的下游。这主要发生在短开放阅读框（ORF）翻译后，而长ORF后的重新启动是罕见的，似乎仅限于几个病毒家族。终止/再循环还与其他两个重要过程相关，即通过No-Go和无义介导的衰变（分别为NGD和NMD）进行的mRNA监视。NGD靶向其上的延伸复合物（EC）通过例如稳定的二级结构而停滞的mRNA。它是通过在停滞的核糖体附近的内切核酸裂解启动的，这是由eRF 1和eRF 3旁系同源物Pelota和Hbs 1刺激的，然后与ABCE 1一起可以在没有预先肽释放的情况下解离EC。NMD靶向含有提前终止密码子（PTC）的异常mRNA，其由异常剪接、突变或转录错误引入。识别和靶向降解含PTC的mRNA是由保守的效应子介导的，包括UPF 1（DEAD盒RNA解旋酶）、UPF 2、UPF 3B和UPF 1激酶SMG 1。UPF 1与eRF 1、eRF 3、UPF 2、UPF 3B和SMG 1相互作用，在该过程中起关键作用。有人建议，核糖体在PTC被逮捕的SURF（SMG 1，UPF 1，eRF 1/3）的监视复合物识别，UPF 1的募集也损害终止。拟议的研究将基于从单个纯化的翻译组分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。在目的1中，我们建议调查的结构，核糖体回收和终止复合物对应的过程中的不同阶段，并表征终止是如何调节顺式作用的mRNA元件和反式作用因子。在目标2中，我们将使用我们的体外重建系统来概括不同的再启动模式，以确定它们的因子需求和机制。在目标3中，我们将定义ABCE 1/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中内切核酸切割的位点并鉴定负责的核酸酶。目的4将致力于调查UPF 1的影响，这取决于其磷酸化状态和UPF 2和UPF 3辅因子的存在，对翻译过程中的各个步骤。