The mechanisms of eukaryotic translation termination and ribosomal recycling

描述(申请人提供)：真核翻译终止由多肽释放因子eRF1和eRF3触发。虽然eRF1识别终止密码子并诱导肽-tRNA的水解，但eRF3的S功能长期以来一直不清楚。最近，我们利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，以及编码四肽和终止密码子的氨基酰tRNAs，重建了真核细胞体外翻译的所有步骤。这使得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。根据这一模型，eRF1、eRF3和GTP与终止前复合体的结合首先引起结构重排，表现为终止前复合体的脚趾两核苷酸前移，导致GTP水解，然后是肽-tRNA的快速水解。ERF1和eRF3之间的协同作用需要通过它们的C-末端结构域直接结合。这项建议的总体目标是进一步表征真核细胞翻译终止的机制，并研究真核细胞蛋白质合成的下一个阶段，即核糖体循环的完全未知的机制。快速动力学技术(激冷流和停流)将被用来确定GTP水解和多肽释放的速率常数，以确定中间步骤(例如，终止复合体的构象变化)并定义它们的动力学，以便建立一个完整的终止动力学框架。我们将用小角X射线和中子散射研究eRF1和eRF3之间的相互作用。尖锐聚焦突变结合详细的功能分析将被用来确定eRF3与eRF1相互作用刺激eRF3 S GTP酶活性及其与GTP结合的机制。为了获得关于终止的全面结构概述，将使用定向紫外光交联和冷冻电子显微镜相结合的方法来确定tRNA、mRNA和两种释放因子的位置，以及SOS核糖体在终止前、终止后和各种终止复合体中的构象状态。我们成功地在体外重建了起始、延长和终止，现在将使我们能够研究蛋白质合成的最后阶段--核糖体循环的机制。为了阐明真核细胞终止后核糖体循环的机制并建立事件的顺序，将确定这一过程中所有步骤(脱酰化tRNA和mRNA的释放，以及808个核糖体解离为亚单位)的因子要求。这种方法将产生真核细胞核糖体循环的第一个模型。过早终止密码子的翻译终止(PSC)是遗传病的常见原因。对终止机制的详细了解将有助于合理开发更有效的PSC抑制治疗药物。

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.