The mechanisms of eukaryotic translation termination and ribosomal recycling

描述（由申请人提供）：真核翻译终止由肽释放因子eRF 1和eRF 3触发。eRF 1识别终止密码子并诱导肽基-tRNA水解，而eRF 3的功能长期以来一直不清楚。我们最近重建的所有步骤的真核生物在体外翻译使用纯化的核糖体亚基，9个起始，2个延伸和2个终止因子和氨酰-tRNA的mRNA编码的四肽，随后终止密码子。这使我们能够提出一种真核生物终止的模型，该模型解释了eRF 1和eRF 3在确保新生多肽快速释放方面的协同作用。根据该模型，eRF 1、eRF 3和GTP与终止前复合物的结合首先诱导结构重排，其表现为归因于终止前复合物的足印的两个核苷酸的前移，这导致GTP水解，随后是肽基-tRNA的快速水解。eRF 1和eRF 3之间的协同作用需要它们通过它们的C-末端结构域直接结合。本提案的总体目标是进一步表征真核生物翻译终止的机制，并研究真核生物蛋白质合成的下一个最后阶段核糖体再循环的完全未知的机制。快速动力学技术（猝灭流和停流）将被应用于确定GTP水解和肽释放的速率常数，以确定中间步骤（例如终止复合物中的构象变化）并定义其动力学，以建立完整的终止动力学框架。eRF 1和eRF 3之间的相互作用将通过小角X射线和中子散射进行研究。将采用精确聚焦的诱变结合详细的功能测定来确定eRF 3与eRF 1的相互作用刺激eRF 3的GTP酶活性及其与GTP结合的机制。为了获得一个全面的结构概述的终止，tRNA，mRNA和两个释放因子的位置，SOS核糖体在终止前，终止后和各种终止复合物的构象状态将使用定向UV交联和冷冻电子显微镜的组合来确定。我们在体外重建起始、延伸和终止的成功，将使我们能够研究蛋白质合成的最后阶段，核糖体再循环的机制。为了描述真核细胞终止后核糖体再循环的机制并建立事件的顺序，将确定该过程中所有步骤（脱酰tRNA和mRNA的释放以及808核糖体解离成亚基）的因子要求。这种方法将产生第一个真核生物核糖体再循环模型。翻译终止于提前终止密码子（PSC）是遗传疾病的常见原因。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.