Protein synthesis on single ribosomes

该研究将在单个核糖体水平上测试mrna定向核糖体蛋白生物合成的关键功能方面。两种新的方法发展，单分子荧光偏振和力反馈红外激光光阱将被应用，提供目前无法获得的关于核糖体延伸周期机制的信息。荧光探针将插入延伸因子（EFs）和转移RNA （tRNA）与已知的，预定的方向。通过研究EFs和tRNA结构变化之间的动力学和角关系，将确定遗传密码的校对和mRNA易位的机制。将阐明EFs是电动机还是开关。tRNA通过核糖体运动的时间将与电场结构变化的时间进行比较。利用光阱，确定了mRNA的机械张力与单个核糖体肽延伸速度（力-速度曲线）之间的关系。改变基质和产物浓度对力速度曲线的影响将用于区分假设的易位机制。蛋白质合成在生物体中无处不在，所有核糖体之间的相似性表明延伸周期是最基本的生物过程之一。因此，在这项应用中，在运动蛋白和核糖体功能、体外单分子力学和新型极化光谱方面具有专业知识的实验室之间的合作将促进对核糖体和g蛋白功能的理解，并将在生物物理学和生物医学领域产生非常广泛的影响。

The research will test crucial functional aspects of mRNA-directed ribosomal protein biosynthesis at the level of individual ribosomes. Two new methodological developments, single molecule fluorescence polarization and force-feedback infrared laser optical trap will be applied, providing information currently unavailable about the mechanism of the ribosomal elongation cycle. Fluorescent probes will be inserted into elongation factors (EFs) and transfer RNA (tRNA) with known, predetermined orientations. The mechanism of proof-reading the genetic code and of translocation along the mRNA will be determined by investigating the kinetics, and angular relationships between, structural changes in EFs and tRNA. Whether EFs are motors or switches will be elucidated. The timing of tRNA motions through the ribosome will be compared to those of structural changes in the EFs. Using the optical trap, the relationship between mechanical tension in mRNA and velocity of peptide elongation (the force-velocity curve) of single ribosomes will be determined. The influence of altering substrate and product concentrations on the force velocity curve will be used in distinguishing hypothetical mechanisms of translocation. Protein synthesis is ubiquitous among living organism and the similarities between all ribosomes indicate that the elongation cycle is one of the most fundamental biological processes. Thus the collaboration proposed in this application between laboratories having expertise in motor protein and ribosome function, in vitro single molecule mechanics, and novel polarization spectroscopy will advance the understanding of ribosomal and G-protein function, and should impact very broadly in biophysics and biomedicine.