Mechanism of Translation Termination

摘要智力优势：在蛋白质合成中，存在于信使RNA （mRNA）序列中的信息通过核糖体在所有生物体中进行的重要过程被翻译成蛋白质。翻译的终止是蛋白质合成的关键步骤，必须以高保真度进行，以防止未完全合成的蛋白质从核糖体中过早释放。释放因子1和2 （RF1和RF2）识别mRNA中的停止密码子，并触发将新完成的蛋白与P位点的tRNA连接的键的水解。本研究的长期目标是研究翻译终止的机制，以确定如何实现高保真度。对与核糖体结合的RF1和RF2晶体结构的研究揭示了RFs中对停止密码子识别至关重要的氨基酸。该项目的第一个目标是探索这些关键残基如何动态地区分有意义和停止密码子。在第二个目标中，该项目将检查RF1和RF2在与核糖体结合时所经历的构象变化的作用。这些构象变化可能作为功能开关，在停止密码子被识别后激活肽基- trna键的水解。目前还没有关于RF1和RF2与核糖体结合时发生的构象变化的信息。在第三个目标中，将阐明释放因子3 （RF3）催化RF1和RF2与核糖体分离的机制。本研究将使用基于荧光的、预稳态动力学分析、关键残基的定点诱变和生化分析来研究翻译终止中的这些突出问题。更广泛的影响：该项目通过整合研究和教学来满足重要的教育需求。少数民族本科生和研究生将在先进生物物理方法方面获得最先进的研究训练。PI积极参与外展活动，如STARS计划和加州大学圣地亚哥分校下一步计划。此外，PI还指导当地高中的学生在实验室进行科学项目。今年夏天，一名来自Canyon Crest Academy的高中生将加入PI的实验室。本科生和高中生将进行RF1和RF2的定点诱变，并协助突变蛋白的纯化和表征，在获得重要科学技能的同时为工作做出贡献。几名本科生在过去做出了重大贡献，并共同撰写了研究出版物。这项研究的结果也将发表在科学期刊上，并广泛传播，以加强对蛋白质合成的科学理解，蛋白质合成是基因表达的核心步骤。

Simpson JosephAbstract Intellectual Merit: In protein synthesis, the information present in messenger RNA (mRNA) sequences is translated into proteins in an essential process carried out by ribosomes in all living organisms. The termination of translation, a crucial step in protein synthesis, must occur with high fidelity, to prevent premature release from the ribosome of incompletely synthesized proteins. Release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the mRNA and trigger hydrolysis of the bond linking the newly completed protein to the tRNA in the P site. The long-term objective of this research is to study the mechanism of translation termination to determine how high fidelity is achieved. Examination of crystal structures of RF1 and RF2 bound to the ribosome has revealed amino acids in the RFs that are crucial for stop codon recognition. The first aim of the project is to probe how these key residues dynamically discriminate between sense and stop codons. In the second aim, the project will examine the roles of conformational changes that RF1 and RF2 undergo when they bind to the ribosome. These conformational changes may serve as functional switches to activate hydrolysis of peptidyl-tRNA linkages after stop codons are recognized. Currently there is no information available about the conformational changes that occur in RF1 and RF2 as they bind to the ribosome. In the third aim, the mechanism by which release factor 3 (RF3) catalyzes the dissociation of RF1 and RF2 from the ribosome will be elucidated. This research will investigate these outstanding questions in translation termination using fluorescence-based, pre-steady state kinetic analysis, site-directed mutagenesis of critical residues, and biochemical assays. Broader Impacts:This project serves an important educational need by integrating research and teaching. Several undergraduate minority students and graduate students will obtain state-of-the-art research training in advanced biophysical methods. The PI actively participates in outreach activities such as the STARS program and the UCSD Next Step program. In addition, the PI has mentored students from the local high schools to carry out science projects in the laboratory. A high school student from Canyon Crest Academy will join the PI's laboratory this summer. The undergraduates and high school student will carry out site-directed mutagenesis of RF1 and RF2 and will also assist in the purification and characterization of mutant proteins, to contribute to the work while gaining important scientific skills. Several undergraduates have contributed significantly in the past and co-authored research publications. The results of this study will also be published in scientific journals and disseminated broadly to enhance scientific understanding of protein synthesis, a central step in gene expression.