Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

翻译动力学：波动在蛋白质合成中的作用

• 批准号: 7223815
• 负责人: Tae-Hee Lee
• 金额: $ 5.94万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7223815
• 关键词: Accounting; Affect; Algorithms; Anticodon; Base Pairing; Cell physiology; Cells; Codon Nucleotides; Communication; Complex; Dissociation; Dyes; EEF1A1 gene; Energy Transfer; Enzymes; Equipment; Excision; Facility Construction Funding Category; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Genetic Transcription; Guanosine Triphosphate; Hydrogen Bonding; Hydrolysis; Individual; Label; Left; Length; Life; Measurement; Measures; Mechanics; Mentors; Messenger RNA; Modeling; Molecular Conformation; Monitor; Motion; Movement; Noise; Object Attachment; Oxygen; Peptide Elongation Factor Tu; Peptides; Phase; Photobleaching; Play; Positioning Attribute; Principal Investigator; Process; Production; Protein Biosynthesis; Proteins; Research; Research Personnel; Resolution; Ribosomes; Role; Signal Transduction; Site; System; Testing; Time; Transfer RNA; Translations; Work; analog; base; cyanine dye 5; data acquisition; gene repair; human EEF1A1 protein; improved; in vivo; instrumentation; programs; protein folding; research study; single molecule; single-molecule FRET; size

DESCRIPTION (provided by applicant): Translation, in vivo protein synthesis, is a vital process in maintaining cell life by producing enzymes performing almost every critical function in the cell including gene transcription, gene repair, protein synthesis, and protein folding/degradation. Understanding translation is essential in controlling cell function/life by offering ways to control enzyme production in the cell. The ribosome selects the correct transfer RNA (tRNA) based on mRNA(codon)-tRNA(anticodon) interaction to synthesize protein with the correct sequence. The selection is composed of two sub-steps--initial selection and proofreading. Candidate is currently trying to elucidate the mechanism of proofreading. During the initial selection, ternary complex of elongation factor Tu (EF-Tu), tRNA, and GTP delivers tRNA to the mRNA/ribosome complex. Only when codon matches with anticodon, EF-Tu hydrolyzes GTP and changes conformation to dissociate from the ribosome. Candidate hypothesizes that this recognition process (codon-dependent GTP hydrolysis on EF-Tu) is enabled by tRNA fluctuations, dynamics of which is determined by codon-anticodon interaction. Therefore, tRNA acts as a communication channel between the ribosome decoding site and EF-Tu. To examine the hypothesis, candidate proposes to monitor individual working ribosome in real-time through single molecule fluorescence measurement. Single molecule measurement enables high time-resolution real-time monitoring of individual steps in non-synchronizable multi-step enzymatic processes. Candidate proposes following specific aims to test the hypothesis: (1) Construct an experimental system to monitor tRNA movement, elongation factor Tu (EF-Tu) movement, and GTP hydrolysis through single molecule fluorescence resonance energy transfer (SM FRET): (a) achieve 3 ms time resolution to monitor the dynamics, (b) label EF-Tu and test fluorescent GTP analogues to monitor EF-Tu movement and GTP hydrolysis; (2) Achieve the highest possible signal-to-noise ratio (S/N) for SM FRET measurements: (a) optimize instrumentation for highest possible S/N, (b) optimize oxygen scavenger system, (c) Implement noise removal algorithm based on stochastic prediction; (3) Relate tRNA motion to GTP hydrolysis and EF-Tu dissociation: (a) monitor GTP hydrolysis and tRNA motion simultaneously, (b) monitor EF-Tu movement and tRNA motion simultaneously. Successful completion of proposed research will greatly enhance our understanding of in translation. Understanding how the translation machinery synthesizes proteins with such an unusually high accuracy will open ways to control cell function/life.

描述（由申请人提供）：翻译，体内蛋白质合成，是维持细胞生命的重要过程，通过产生执行细胞中几乎所有关键功能的酶，包括基因转录，基因修复，蛋白质合成和蛋白质折叠/降解。通过提供控制细胞中酶产生的方法，了解翻译对于控制细胞功能/生命至关重要。核糖体根据mRNA（密码子）-tRNA（反密码子）相互作用选择正确的转运RNA（tRNA）合成具有正确序列的蛋白质。选择由两个子步骤组成——初步选择和校对。候选人目前正试图阐明校对的机制。在初始选择过程中，延伸因子Tu （EF-Tu）、tRNA和GTP的三元配合物将tRNA传递给mRNA/核糖体复合物。只有当密码子与反密码子匹配时，EF-Tu才能水解GTP并改变构象与核糖体分离。候选人假设这种识别过程（密码子依赖于EF-Tu上的GTP水解）是由tRNA波动激活的，其动力学是由密码子-反密码子相互作用决定的。因此，tRNA作为核糖体解码位点与EF-Tu之间的通信通道。为了验证这一假设，候选人建议通过单分子荧光测量实时监测单个工作核糖体。单分子测量能够对非同步多步骤酶促过程中的单个步骤进行高时间分辨率实时监测。