Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

翻译，在体内蛋白质合成，是维持细胞生命的重要过程，通过产生酶执行 细胞中几乎所有的关键功能，包括基因转录、基因修复、蛋白质合成和蛋白质 折叠/降解。理解翻译是控制细胞功能/生命的关键， 控制细胞内酶的产生。核糖体选择正确的转运RNA（tRNA）， mRNA（密码子）-tRNA（反密码子）相互作用以合成具有正确序列的蛋白质。这项选择是 由两个子步骤组成-初始选择和校对。候选人目前正试图阐明 校对机制。在最初的选择过程中，延伸因子Tu（EF-Tu），tRNA， GTP将tRNA传递给mRNA/核糖体复合物。仅当密码子与反密码子匹配时，EF-Tu 水解GTP并改变构象以从核糖体解离。候选人假设， 识别过程（EF-Tu上的密码子依赖性GTP水解）通过tRNA波动实现， 其动力学由密码子-反密码子相互作用决定。因此，tRNA作为 核糖体解码位点和EF-Tu之间的通讯通道。为了验证这个假设， 候选人建议通过单分子荧光实时监测单个工作核糖体 测量.单分子测量能够实现高时间分辨率实时监测 非同步多步酶促过程中的各个步骤。候选人建议如下 具体目的是检验假设：#1构建一个实验系统来监测tRNA运动， 延伸因子Tu（EF-Tu）运动和GTP水解通过单分子荧光共振 能量转移（SM FRET）1）实现3 ms的时间分辨率，以监测动力学，2）标记EF-Tu并测试 荧光GTP类似物，用于监测EF-Tu运动和GTP水解，#2 SM FRET测量的信噪比（S/N）1）优化仪器以获得最高可能的S/N，2） 优化除氧系统，3）实现基于随机预测的噪声去除算法，#3 将tRNA运动与GTP水解和EF-Tu解离相关联1）监测GTP水解和tRNA运动 2）同时监测EF-Tu运动和tRNA运动。成功完成 本文的研究将大大提高我们对翻译的理解。理解翻译如何 机器以如此高的准确度合成蛋白质，将为控制细胞生长开辟道路。 功能/寿命。

Translation, in vivo protein synthesis, is 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) 1) Achieve 3 ms time resolution to monitor the dynamics, 2) 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) forSM FRET measurements 1) Optimize instrumentation for highest possible S/N, 2) Optimize oxygen scavenger system, 3) Implement noise removal algorithm based on stochastic prediction, #3 Relate tRNA motion to GTP hydrolysis and EF-Tu dissociation 1) Monitor GTP hydrolysis and tRNA motion simultaneously, 2) Monitor EF-Tu movement and tRNA motion simultaneously. Successful completion of proposed research will greatly enhance our understandings in translation. Understanding how the translation machinery synthesizes proteins with such an unusually high accuracy will open ways to control cell function/life.