Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

翻译，即体内蛋白质的合成，是产生几乎具有以下功能的酶的重要细胞过程 细胞中的每一项关键功能。这些功能包括基因转录、基因修复、蛋白质合成、 和蛋白质折叠/降解。因此，理解翻译对于控制细胞功能是至关重要的。 核糖体根据mRNA(密码子)-tRNA(反密码子)的相互作用选择正确的转移RNA(TRNA)以 用正确的序列合成蛋白质。选择由两个子步骤组成--初始选择 还有校对。侯选人提出研究的目的是为了阐明最初的 选择。在初始选择过程中，延伸因子Tu(EF-Tu)、tRNA和GTP的三元复合体 将tRNA运送到核糖体。只有当密码子与反密码子匹配时，EF-Tu才能水解GTP和 改变其构象，使其与核糖体解离。候选人假设依赖于密码子的 Ef-Tu上的GTP水解酶是由核糖体诱导的tRNA运动提供能量的 构象变化。核糖体在三元结合时的构象变化 复合体依赖于密码子-反密码子的相互作用。因此，tRNA起到了通讯通道的作用 在核糖体和EF-Tu之间。为了检验这一假设，候选人建议监控个人的工作情况 通过单分子荧光技术实时检测核糖体。单分子方法使 非同步多步中单个步的高时间分辨率实时监控 酶促过程。候选人提出了以下具体目标来检验假设：#1构建一个 监测tRNA运动、延伸因子Tu(EF-Tu)运动和GTP水解度的实验系统 事件通过单分子荧光技术1)达到3ms的时间分辨率进行监测 荧光强度的变化，2)荧光标记EF-Tu而不干扰其内部序列，以及3) 测试荧光GTP类似物以同时监控EF-Tu运动和GTP水解，#2实现 单分子荧光共振能量转移的最高可能信噪比(S/N) 测量1)优化仪器以获得尽可能高的S/N，2)优化除氧器系统，3) 改进了基于隐马尔可夫模型的数据分析算法；4)设置了单光子计数 系统，#3将tRNA运动与GTP水解和EF-Tu解离联系起来1)监测GTP水解和 2)同时监测EF-Tu运动和tRNA运动。成功 完成拟议的研究将增强我们对翻译机制如何 实现高精度的蛋白质合成。

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.