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: 7458984
• 负责人: Tae-Hee Lee
• 金额: $ 24.22万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 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 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458984
• 关键词: Accounting; Algorithms; Anticodon; Binding; Cell physiology; Cells; Charge; Codon Nucleotides; Communication; Complement; Complex; Count; Data; Data Analyses; Databases; Devices; Dissociation; Dyes; EEF1A1 gene; Enzymes; Equipment; Event; Facility Construction Funding Category; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Genetic Transcription; Guanosine Triphosphate; Hydrolysis; Image; Individual; Kinetics; Label; Length; Life; Measurement; Measures; Mechanics; Messenger RNA; Methods; Molecular Conformation; Monitor; Motion; Movement; Noise; Oxygen; Peptide Elongation Factor Tu; Phase; Photobleaching; Photons; Play; Process; Protein Biosynthesis; Proteins; Purpose; Research; Resolution; Ribosomes; Role; Signal Transduction; Site; System; Techniques; Testing; Time; Transfer RNA; Translations; Work; analog; base; cyanine dye 5; data acquisition; gene repair; human EEF1A1 protein; improved; in vivo; instrument; markov model; protein folding; research study; single molecule; single-molecule FRET; size

Translation, in vivo protein synthesis, is a vital cellular process to produce enzymes that perform almost every critical function in the cell. These functions include gene transcription, gene repair, protein synthesis, and protein folding/degradation. Therefore, understanding translation is essential in controlling cell function. The ribosome selects correct transfer RNA (tRNA) based on mRNA(codon)-tRNA(anticodon) interaction to synthesize proteins with correct sequences. The selection is composed of two sub-steps - initial selection and proofreading. The purpose of candidate's proposed research is to elucidate the mechanism of the initial selection. During the initial selection, ternary complex of elongation factor Tu (EF-Tu), tRNA, and GTP delivers tRNA to the ribosome. Only when codon matches with anticodon, EF-Tu hydrolyzes GTP and changes its conformation to dissociate from the ribosome. Candidate hypothesizes that the codondependent GTP hydrolysis on EF-Tu is energized by tRNA motion, which is induced by the ribosomal conformational change. The conformational change of the ribosome upon the binding of the ternary complex depends on codon-anticodon interaction. Therefore, tRNA acts as a communication channel between the ribosome and EF-Tu. To test the hypothesis, candidate proposes to monitor individual working ribosome in real-time through single molecule fluorescence techniques. Single molecule methods enable 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 motion, elongation factor Tu (EF-Tu) movement, and GTP hydrolysis event through single molecule fluorescence techniques 1) Achieve 3 ms time resolution to monitor fluorescence intensity changes, 2) Fluorescently label EF-Tu without disturbing its internal sequence, and 3) test fluorescent GTP analogues to monitor EF-Tu movement and GTP hydrolysis simultaneously, #2 Achieve the highest possible signal to noise ratio (S/N) for single molecule fluorescence resonance energy transfer measurements 1) Optimize instruments for highest possible S/N, 2) Optimize oxygen scavenger system, 3) Improve data analysis algorithm based on hidden Markov models, and 4) Setup a single photon counting system, #3 Relate tRNA motion to GTP hydrolysis and EF-Tu dissociation 1) Monitor GTP hydrolysis and tRNA motion simultaneously, and 2) Monitor EF-Tu movement and tRNA motion simultaneously. Successful completion of proposed research will enhance our understandings about how the translation machinery achieves high accuracy protein synthesis.

翻译，在体内蛋白质合成，是一个重要的细胞过程，产生的酶几乎执行