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。它包括至少两个基本功能，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eRF 1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。II类释放因子eRF 3以GTP依赖性方式协助终止过程。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性过早终止突变的治疗策略。eRF 1蛋白包含三个离散结构域。结构和遗传数据的考虑导致了以下建议：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释放;结构域3介导eRF 1与eRF 3之间的相互作用。竞争模型认为，TASNIKS基序或结构域1中的YCF基序对终止密码子识别至关重要。本提案的第一个目的是确定参与终止密码子识别的eRF 1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SUP 45基因编码eRF 1。我们最近发现，SUP 45 mRNA的半衰期是由终止过程的效率调节的。当终止受到损害时，这种机制导致eRF 1蛋白水平增加。本提案的第二个目的将测试该模型，并探索这种新的调控机制如何控制SUP 45 mRNA和eRF 1蛋白水平。我们最近发现，以前未表征的蛋白Tpa 1 p影响翻译终止的效率，mRNA poly（A）尾长，和mRNA在酵母细胞中的半衰期。这使我们提出了一个模型，其中Tpa 1 p耦合翻译终止细胞mRNA的去腺苷酸化。本提案的第三个目的是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.