Mechanism of Translation Termination

翻译终止机制

• 批准号: 1158127
• 负责人: Simpson Joseph
• 金额: $ 60万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158127&HistoricalAwards=false
• 关键词: Mechanism; Translation; Termination

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.

在蛋白质合成中，信使RNA（mRNA）序列中的信息在所有生物体中的核糖体进行的一个重要过程中被翻译成蛋白质。翻译的终止是蛋白质合成的关键步骤，必须以高保真度发生，以防止不完全合成的蛋白质从核糖体中过早释放。 释放因子1和2（RF 1和RF 2）识别mRNA中的终止密码子，并触发将新完成的蛋白质连接到P位点的tRNA的键的水解。 本研究的长期目标是研究翻译终止的机制，以确定如何达到高保真度。 结合到核糖体的RF 1和RF 2的晶体结构的检查已经揭示了RF中对于终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区分有义密码子和终止密码子。 在第二个目标中，该项目将研究RF 1和RF 2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，激活水解后终止密码子识别的肽基-tRNA键。 目前还没有关于RF 1和RF 2与核糖体结合时发生的构象变化的信息。 在第三个目的中，将阐明释放因子3（RF 3）催化RF 1和RF 2从核糖体解离的机制。 本研究将使用基于荧光的预稳态动力学分析、关键残基的定点突变和生化测定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。 一些本科少数民族学生和研究生将获得先进的生物物理方法的最先进的研究培训。 PI积极参与外展活动，如STARS计划和UCSD下一步计划。 此外，PI还指导当地高中的学生在实验室开展科学项目。 今年夏天，一名来自峡谷佳洁士学院的高中生将加入PI的实验室。 本科生和高中生将进行RF 1和RF 2的定点诱变，并将协助纯化和表征突变蛋白，为这项工作做出贡献，同时获得重要的科学技能。 一些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，以提高对蛋白质合成的科学理解，这是基因表达的核心步骤。