Mechanism of Eukaryotic Translation Termination

描述(申请人提供)：翻译终止是蛋白质合成的最后阶段。它至少包括两个基本功能，即终止密码子识别和多肽链释放。在真核生物中，I类释放因子eRF1识别三个终止密码子(UAA、UAG和UGA)中的每一个，并介导新生多肽链的释放。第二类释放因子eRF3以依赖于GTP的方式辅助终止过程。该项目的长期目标是更好地了解翻译终止的过程，以便开发旨在抑制致病过早停止突变的治疗策略。ERF1蛋白包含三个不同的结构域。对结构和遗传数据的考虑导致了以下假设：结构域1介导终止密码子识别；结构域2与核糖体的肽基转移酶中心相互作用以促进多肽链的释放；结构域3介导eRF1与eRF3之间的相互作用。相互竞争的模型认为，结构域1中的TASNIKS基序或YCF基序对于终止密码子识别至关重要。这项建议的第一个目的是确定参与终止密码子识别的eRF1结构域1的关键残基，以测试这些竞争模型的相对优点。酵母SUP45基因编码eRF1。我们最近发现，SUP45 mRNA的半衰期受终止过程的效率调节。当终止受到损害时，这种机制会导致eRF1蛋白水平的增加。这项提案的第二个目的将测试这一模型，并探索这一新的调控机制如何控制SUP45 mRNA和eRF1蛋白水平。我们最近发现，以前未知的蛋白Tpa1p影响酵母细胞的翻译终止效率、mRNA聚(A)尾长和mRNA半衰期。这导致我们提出了一个模型，在该模型中，Tpa1p将翻译终止与细胞mRNAs的去烯化结合在一起。该提案的第三个目标将测试该模型的各个方面，以便我们能够更好地理解翻译终止和mRNA稳定性之间的重要相互作用。

DESCRIPTION (provided by applicant): Translation termination is the final stage of protein synthesis. It includes at least two essential functions, stop codon recognition and polypeptide chain release. In eukaryotic organisms, the class I release factor eRF1 recognizes each of the three termination codons (UAA, UAG, and UGA) and mediates release of the nascent polypeptide chain. The class II release factor eRF3 assists the termination process in a GTP-dependent manner. The long-term goal of this project is to better understand the process of translation termination so therapeutic strategies aimed at the suppression of disease-causing premature stop mutations can be developed. The eRF1 protein contains three discrete domains. A consideration of structural and genetic data led to the proposal that domain 1 mediates stop codon recognition; domain 2 interacts with the peptidyl transferase center of the ribosome to facilitate polypeptide chain release; and domain 3 mediates the interaction between eRF1 with eRF3. Competing models argue that either the TASNIKS motif or the YCF motif in domain 1 is critical for stop codon recognition. The first aim of this proposal will identify key residues of eRF1 domain 1 involved in stop codon recognition to test the relative merits of these competing models. The yeast SUP45 gene encodes eRF1. We recently discovered that the half-life of the SUP45 mRNA is regulated by the efficiency of the termination process. This mechanism leads to an increase in the eRF1 protein level when termination is compromised. The second aim of this proposal will test this model and explore how this novel regulatory mechanism controls SUP45 mRNA and eRF1 protein levels. We recently found that the previously uncharacterized protein Tpa1p influences the efficiency of translation termination, mRNA poly(A) tail length, and mRNA half-life in yeast cells. This led us to propose a model in which Tpa1p couples translation termination to the deadenylation of cellular mRNAs. The third aim of this proposal will test various aspects of this model so we can better understand the important interplay between translation termination and mRNA stability.