The mechanisms of eukaryotic translation termination and ribosomal recycling

真核生物翻译终止和核糖体回收的机制

• 批准号: 7808758
• 负责人: TATYANA V PESTOVA
• 金额: $ 29.34万
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808758
• 关键词: Accounting; Binding; Biological Assay; C-terminal; Complex; Cryoelectron Microscopy; DNA Sequence Rearrangement; Data; Development; Dissociation; Ensure; Esters; Eukaryota; Event; GTP Binding; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Homologous Gene; Hydrolysis; In Vitro; Individual; Ions; Kinetics; Mediating; Messenger RNA; Modeling; Molecular Conformation; Mutagenesis; Neutrons; Nonsense Codon; Nucleotides; Peptide Initiation Factors; Peptides; Peptidyltransferase; Positioning Attribute; Printing; Process; Prokaryotic Cells; Protein Biosynthesis; Recycling; Research Personnel; Ribosomes; Roentgen Rays; Role; Site; Solutions; Son of Sevenless Proteins; Staging; Structure; Techniques; Terminator Codon; Testing; Toes; Transfer RNA; Translations; Work; crosslink; peptidyl-tRNA; polypeptide; reconstitution; release factor; ribosome releasing factor; stopped-flow fluorescence; success; termination factor; translation factor

DESCRIPTION (provided by applicant): Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes the stop codon and induces hydrolysis of peptidyl-tRNA, eRF3's function has for long been obscure. We recently reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits, 9 initiation, 2 elongation and 2 termination factors and aminoacyl-tRNAs on mRNA encoding a tetrapeptide followed by a stop codon. This allowed us to propose a model for eukaryotic termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. According to this model, binding of eRF1, eRF3 and GTP to pre-termination complexes first induces a structural rearrangement, which is manifested as a two-nucleotide forward shift of the toeprint attributed to pre-termination complexes, that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl-tRNA. Cooperativity between eRF1 and eRF3 requires their direct binding through their C-terminal domains. The overall objective of this proposal is to further characterize the mechanism of eukaryotic translation termination and to investigate the completely unknown mechanism of the next, final stage of eukaryotic protein synthesis, ribosomal recycling. Fast kinetics techniques (quench-flow and stopped-flow) will be applied to determine rate constants for GTP hydrolysis and peptide release, to identify intermediate steps (e.g. conformational changes in termination complexes) and to define their kinetics in order to establish a complete kinetic frame-work of termination. The interaction between eRF1 and eRF3 will be studied by small-angle X-ray and neutron scattering. Sharply focused mutagenesis combined with detailed functional assays will be employed to determine the mechanism, by which interaction of eRF3 with eRF1 stimulates eRF3 s GTPase activity and its binding to GTP. To obtain a comprehensive structural overview of termination, the positions of tRNA, mRNA and both release factors, and conformational states of SOS ribosomes in pre-termination, post-termination and various termination complexes will be determined using a combination of directed UV cross-linking and cryo-electron microscopy. Our success in reconstituting in vitro initiation, elongation and termination will now allow us to investigate the mechanism of the last stage of protein synthesis, ribosomal recycling. To delineate the mechanism of eukaryotic post-termination ribosomal recycling and to establish the order of events, the factor requirements for all steps in this process (release of deacylated tRNA and mRNA, and dissociation of 808 ribosomes into subunits) will be determined. This approach will yield the first model of eukaryotic ribosomal recycling. Translation termination at premature stop codon (PSC) is a frequent cause of genetic disease. Detailed understanding of the mechanism of termination will facilitate rational development of more efficacious agents for PSC suppression therapy.

描述（由申请人提供）：真核翻译终止由肽释放因子eRF1和eRF3触发。 eRF1 识别终止密码子并诱导肽基-tRNA 水解，而 eRF3 的功能长期以来一直不清楚。我们最近使用纯化的核糖体亚基、9 个起始因子、2 个延伸因子和 2 个终止因子以及编码四肽后跟终止密码子的 mRNA 上的氨酰基-tRNA 在体外重建了真核翻译的所有步骤。这使我们能够提出一个真核终止模型，该模型解释了 eRF1 和 eRF3 在确保新生多肽快速释放方面的协同作用。根据该模型，eRF1、eRF3 和 GTP 与终止前复合物的结合首先诱导结构重排，这表现为归因于终止前复合物的趾纹向前移动两个核苷酸，导致 GTP 水解，随后肽基-tRNA 快速水解。 eRF1 和 eRF3 之间的协同作用需要它们通过 C 端结构域直接结合。该提案的总体目标是进一步表征真核翻译终止的机制，并研究真核蛋白质合成的下一个、最后阶段——核糖体回收的完全未知的机制。快速动力学技术（淬灭流和停流）将用于确定 GTP 水解和肽释放的速率常数，识别中间步骤（例如终止复合物中的构象变化）并定义其动力学，以便建立完整的终止动力学框架。 eRF1 和 eRF3 之间的相互作用将通过小角 X 射线和中子散射来研究。将采用针对性强的诱变结合详细的功能测定来确定 eRF3 与 eRF1 相互作用刺激 eRF3 GTP 酶活性及其与 GTP 结合的机制。为了获得终止的全面结构概览，将使用定向紫外交联和冷冻电子显微镜相结合来确定终止前、终止后和各种终止复合物中 tRNA、mRNA 和两种释放因子的位置以及 SOS 核糖体的构象状态。我们在体外重建起始、延伸和终止方面的成功将使我们能够研究蛋白质合成最后阶段、核糖体回收的机制。为了描述真核细胞终止后核糖体回收的机制并确定事件的顺序，将确定该过程中所有步骤的因子要求（脱酰化 tRNA 和 mRNA 的释放，以及 808 核糖体解离为亚基）。这种方法将产生第一个真核核糖体回收模型。过早终止密码子 (PSC) 处的翻译终止是遗传病的常见原因。详细了解终止机制将有助于合理开发更有效的 PSC 抑制治疗药物。