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）：真核翻译终止是由肽释放因子ERF1和ERF3触发的。尽管ERF1识别终止密码子并诱导肽基TRNA的水解，但ERF3的功能长期存在。最近，我们使用纯化的核糖体亚基，9个起始，2个伸长率和2个终止因子和2个终止因子以及编码四肽，然后是停止密码子的mRNA，在体外重新建立了真核翻译的所有步骤。这使我们能够提出一个真核终止的模型，该模型解释了ERF1和ERF3在确保快速释放新生多肽时的合作作用。根据该模型，ERF1，ERF3和GTP对预终止络合物的结合首先诱导结构重排，这表现为归因于前末端复合物的两核苷酸向前移位，这导致了GTP水解，从而导致肽基水解的快速水解肽基 - 肽基 - 肽基。 ERF1和ERF3之间的合作性需要通过其C末端域的直接结合。该提案的总体目的是进一步表征真核翻译终止的机制，并研究下一个真核蛋白质合成的最后阶段的完全未知的机制，核糖体回收。快速动力学技术（Quench-Flow and oferped-flow）将用于确定GTP水解和肽释放的速率常数，以识别中间步骤（例如，终止复合物的构象变化）并定义其动力学以建立终端动力学框架。 ERF1和ERF3之间的相互作用将通过小角度X射线和中子散射研究。将使用浓缩的诱变与详细的功能测定法相结合来确定机制，通过该机制，ERF3与ERF1的相互作用刺激ERF3 S GTPase活性及其与GTP的结合。为了获得终止的全面结构概述，将使用有向UV的交联和冷冻链接显微镜的组合确定tRNA，mRNA和两个释放因子的位置以及SOS核糖体的两个释放因子以及SOS核糖体的构象状态。我们在重建体外开始，伸长和终止方面的成功现在将使我们能够研究蛋白质合成的最后阶段的机理，核糖体回收。为了描述真核后末端核糖体回收的机制并确定事件的顺序，将确定此过程中所有步骤的因子要求（释放了脱酰化的tRNA和mRNA，以及将808个核糖体解离为亚基）。这种方法将产生第一个真核核糖体回收模型。过早停止密码子（PSC）的翻译终止是遗传疾病的常见原因。对终止机制的详细理解将有助于合理地发展更有效的PSC抑制疗法。