Protein synthesis on single ribosomes

该研究将在单个核糖体水平上测试mRNA指导的核糖体蛋白生物合成的关键功能方面。 两个新的方法的发展，单分子荧光偏振和力反馈红外激光光阱将被应用，提供信息目前无法获得的核糖体延长周期的机制。 荧光探针将以已知的预定方向插入延伸因子（EF）和转移RNA（tRNA）中。 通过研究EFs和tRNA的动力学和结构变化之间的角度关系，将确定遗传密码的校对机制和mRNA的沿着易位机制。将阐明EF是电动机还是开关。 tRNA在核糖体中运动的时间将与EF中结构变化的时间进行比较。 使用光阱，将确定mRNA中的机械张力与单个核糖体的肽延伸速度（力-速度曲线）之间的关系。 改变底物和产物浓度对力-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普遍存在，所有核糖体之间的相似性表明延伸周期是最基本的生物学过程之一。 因此，在本申请中提出的合作实验室之间的专业知识，在电机蛋白和核糖体功能，体外单分子力学，和新的偏振光谱将推进核糖体和G蛋白功能的理解，并应在生物物理学和生物医学的影响非常广泛。

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.