Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

翻译，即体内蛋白质的合成，是通过产生酶来维持细胞生命的重要过程 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。了解翻译对控制细胞功能/寿命至关重要，因为它提供了 控制细胞中的酶的产生。核糖体根据以下条件选择正确的转移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。可选的是 由两个子步骤组成--初选和校对。候选人目前正试图澄清 校对机制。在初始选择过程中，伸长因子Tu(EF-Tu)、tRNA、 而GTP将tRNA递送到mRNA/核糖体复合体。仅当密码子与反密码子匹配时，EF-Tu 水解GTP并改变构象使其从核糖体解离。候选人假设这一点 识别过程(依赖于密码子的GTP对EF-Tu的水解)由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作为一种 核糖体解码位点和EF-Tu之间的通讯通道。为了检验这一假设， 候选人提出通过单分子荧光实时监测单个工作核糖体 测量。单分子测量使高时间分辨率的实时监测成为可能 在不可同步的多步骤酶过程中的各个步骤。候选人建议遵循以下原则 具体目的是验证这一假设：#1构建一个实验系统来监测tRNA的运动， 延伸因子Tu(EF-Tu)运动和GTP单分子荧光共振水解性研究 能量传输(SM FRET)1)达到3ms的时间分辨率以监测动态，2)标记EF-Tu并进行测试 荧光GTP类似物用于监测EF-Tu运动和GTP水解，#2实现了最高可能 SM FRET测量的信噪比(S/N)1)优化仪器以获得尽可能高的S/N，2) 优化除氧器系统，3)实现基于随机预测的噪声去除算法，#3 TRNA运动与GTP水解和EF-Tu解离的关系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 提出的研究将极大地提高我们对翻译的理解。了解翻译是如何 机器以如此高的精度合成蛋白质将打开控制细胞的方法 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.