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: 8372177
• 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/8372177
• 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; 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. PUBLIC HEALTH RELEVANCE: Mutations in the translation apparatus leading to defects in stop codon recognition and uORF-mediated translational control are implicated in genetic diseases, e.g. cystic fibrosis, muscular dystrophy, hereditary thrombocythemia and many others. About a third of all inherited human disorders are caused by mutations that introduce premature termination codons (PTC), which trigger mRNA degradation by nonsense-mediated decay (NMD). Clinically relevant antibiotics suppress PTC recognition, enhancing readthrough and ameliorating disease by partially restoring synthesis of full-length protein and impairing NMD, but the efficacy of suppression is variable: more detailed characterization of termination and NMD would facilitate further rational development of PTC suppression therapy.)

描述（由申请人提供）：真核终止是eRF1和eRF3两种释放因子之间复杂的功能相互作用的结果，其中eRF3对GTP的水解与eRF1对肽基trna的水解相互作用。终止后复合物（post-TCs）的再循环是由ABCE1和eRF1共同作用促进的，ABCE1是atp结合盒蛋白家族的重要成员，eRF1在肽释放后仍与后TCs相关。因此，eRF1参与了蛋白质合成、终止和核糖体再循环两个连续的阶段。在某些情况下，后tcs不进行完全的再循环，而终止之后是在相同的mRNA上重新启动，通常在停止密码子的下游。这主要发生在翻译短的开放阅读框（orf）后，而在长orf后重新启动是罕见的，似乎仅限于几个病毒科。终止/再循环还与另外两个重要的过程有关，即No-Go和nonsense介导的衰变（分别为NGD和NMD）对mRNA的监视。NGD靶向的mrna上的延伸复合物（ECs）被稳定的二级结构所阻滞。它是由停止的核糖体附近的核内溶分裂引起的，这是由eRF1和eRF3的旁系物Pelota和Hbs1刺激的，然后与ABCE1一起可以解离ECs，而无需事先释放肽。NMD针对的是由异常剪接、突变或转录错误引入的含有过早终止密码子（ptc）的异常mrna。含有ptc的mrna的识别和靶向降解是由保守效应物介导的，包括UPF1（一种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，在这一过程中起关键作用。研究表明，在ptc上捕获的核糖体被SURF （SMG1, UPF1, eRF1/3）监视复合体识别，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组分中体外重建蛋白质合成的所有阶段（起始，延伸，终止和核糖体循环）的方法。在Aim 1中，我们建议研究核糖体循环和终止复合物的结构，对应于该过程的不同阶段，并表征终止是如何由顺式作用的mRNA元件和反式作用的因子调节的。在目标2中，我们将概述使用体外重构系统重新启动的不同模式，以确定其因子需求和机制。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中核内溶解裂解的位置，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，这取决于其磷酸化状态和UPF2和UPF3辅因子的存在。