Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

翻译，在体内蛋白质合成，是一个重要的细胞过程，产生酶，执行几乎 细胞中的每一个关键功能。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至关重要。 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）， 合成具有正确序列的蛋白质。该选择由两个子步骤组成-初始选择 还有校对候选人提出的研究目的是阐明初始 选择.在最初的选择过程中，延伸因子Tu（EF-Tu）、tRNA和GTP的三元复合物 将tRNA传递给核糖体。只有当密码子与反密码子匹配时，EF-Tu才水解GTP， 改变其构象以从核糖体解离。候选人假设密码子依赖性 EF-Tu上的GTP水解是由tRNA运动激发的，tRNA运动是由核糖体诱导的。 构象变化核糖体在三聚体结合时的构象变化 复合物依赖于密码子-反密码子相互作用。因此，tRNA作为一种通讯渠道， 核糖体和EF-Tu之间。为了验证假设，候选人建议监控个人工作 通过单分子荧光技术实时检测核糖体。单分子方法使 非同步多步骤中单个步骤的高时间分辨率实时监测 酶促过程。候选人提出以下具体目标来测试假设：#1构建一个 监测tRNA运动、延伸因子Tu（EF-Tu）运动和GTP水解的实验系统 1）实现3 ms的时间分辨率，以监测 荧光强度变化，2）荧光标记EF-Tu而不干扰其内部序列，和3） 测试荧光GTP类似物，同时监测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） 1）优化仪器以获得尽可能高的S/N，2）优化除氧剂系统，3） 改进基于隐马尔可夫模型的数据分析算法; 4）建立单光子计数 系统，#3将tRNA运动与GTP水解和EF-Tu解离相关联1）监测GTP水解， 同时监测tRNA运动和EF-Tu运动。成功 完成拟议的研究将提高我们的理解如何翻译机制 实现高精度蛋白质合成。

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.