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）：真核终止是由两个释放因子ERF1和ERF3之间的复杂功能相互作用引起的，其中ERF3夫妇通过ERF1通过ERF3夫妇密码子识别和肽基TRNA水解进行了GTP水解。 ABCE1的协同作用是ABCE1的协同作用（ATP结合盒子的蛋白质的基本成员）和ERF1的回收促进了末端终端复合物（TCS后）的回收，而ERF1的基本成员仍然与肽释放后与后TC相关联。因此，ERF1参与了蛋白质合成，终止和核糖体回收的连续两个阶段。在某些情况下，TC后不会进行完全的回收利用，而是终止是对同一mRNA（通常是终止密码子的下游）重新定期。这主要发生在简短的开放式阅读框（ORF）翻译后，而长ORF后重新定期很少，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的过程相关，即通过No-Go和废话介导的衰减（NGD和NMD）进行的mRNA监测。 NGD靶标mRNA，其伸长率复合物（EC）的靶标被例如稳定的二级结构。它是由停滞核糖体附近的内核酸裂解引发的，ERF1和ERF3副同源物Pelota和Hbs1刺激了它，然后与ABCE1一起可以在没有事先肽释放的情况下与ECS解离ECS。 NMD靶标​​的异常mRNA包含过早终止密码子（PTC），这些mRNA是通过异常的剪接，突变或转录误差引入的。识别和靶向含PTC的含PTC的mRNA的靶向是由保守的效应子介导的，包括UPF1（Dead-Box RNA Helicase），UPF2，UPF3B和UPF1激酶SMG1。 UPF1与ERF1，ERF3，UPF2，UPF3B和SMG1相互作用，并在此过程中起关键作用。有人提出，在PTC中捕获的核糖体被冲浪（SMG1，UPF1，ERF1/3）监测复合体认识到，并且UPF1的募集也损害了终止。拟议的研究将基于从单个纯化的翻译成分中的蛋白质合成（启动，伸长，终止和核糖瘤回收）的体外重构的方法。在AIM 1中，我们建议研究与该过程中不同阶段相对应的核糖体回收和终止复合物的结构，并表征如何通过顺式作用mRNA元素和反式作用因子来调节终止。在AIM 2中，我们将使用我们的体外重组系统来概括不同的重新定制模式，以确定其因素要求和机制。在AIM 3中，我们将定义ABCE1/Pelota介导的核糖体伸长复合物的解离，并将尝试将NGD中核核酸裂解部位定位并识别负责任的核酸酶。 AIM 4将专门研究UPF1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，对翻译过程中的各个步骤。 公共卫生相关性：翻译机构中的突变导致停止密码子识别和UORF介导的翻译控制中的缺陷与遗传疾病有关，例如囊性纤维化，肌肉营养不良，遗传性血小板血症等。大约三分之一的遗传性人类疾病是由引入过早终止密码子（PTC）引起的突变引起的，该突变引起了无义介导的衰减（NMD）触发mRNA降解。临床相关的抗生素抑制PTC识别，通过部分恢复全长蛋白质和NMD损害的合成来抑制PTC识别，并改善疾病，但抑制的功效是可变的：终止和NMD的详细表征和NMD可以促进PTC抑制疗法的进一步发展。）