Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

描述（由申请人提供）：翻译，即体内蛋白质合成，是维持细胞生命的重要过程，通过产生在细胞中执行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。理解翻译是通过提供控制细胞中酶产生的方法来控制细胞功能/生命的关键。核糖体基于mRNA（密码子）-tRNA（反密码子）相互作用选择正确的转运RNA（tRNA）以合成具有正确序列的蛋白质。选书分为初选和校对两个步骤。候选人目前正试图阐明校对的机制。在初始选择期间，延伸因子Tu（EF-Tu）、tRNA和GTP的三元复合物将tRNA递送至mRNA/核糖体复合物。只有当密码子与反密码子匹配时，EF-Tu才水解GTP并改变构象从核糖体上解离。候选人假设这种识别过程（EF-Tu上的密码子依赖性GTP水解）是由tRNA波动实现的，其动力学由密码子-反密码子相互作用决定。因此，tRNA充当核糖体解码位点和EF-Tu之间的通信通道。为了检验这一假设，候选人提议通过单分子荧光测量实时监测单个工作核糖体。单分子测量可实现对非同步多步酶促过程中各个步骤的高时间分辨率实时监测。 候选人提出了以下具体目标来验证假设：（1）构建一个通过单分子荧光共振能量转移（SM FRET）监测tRNA运动、延伸因子Tu（EF-Tu）运动和GTP水解的实验系统：（a）达到3 ms的时间分辨率来监测动力学，（B）标记EF-Tu并测试荧光GTP类似物来监测EF-Tu运动和GTP水解;（2）实现SM FRET测量的最高可能信噪比（S/N）：（a）优化仪器以获得最高可能S/N，（B）优化氧清除剂系统，（c）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：（a）同时监测GTP水解和tRNA运动，（B）同时监测EF-Tu运动和tRNA运动。成功地完成拟议的研究将大大提高我们对翻译的理解。了解翻译机制如何以如此高的准确度合成蛋白质，将为控制细胞功能/生命开辟道路。

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.