Ribosome Structure and Function

项目摘要 我们正在进入一个新的和令人兴奋的核糖体研究的时代，其中的作用， 蛋白质合成中的核糖体动力学开始出现。该提案重点 关于核糖体结构动力学的作用，特别是核糖体RNA， 翻译的机制。我们的方法结合了生物化学， 生物物理学、遗传学和晶体学。一个主要目标是阐明 mRNA和tRNA的偶联易位。一种策略是执行一项 详尽的筛选延伸因子EF-G的显性致死突变，我们 预测将在先前未观察到的瞬时易位状态中捕获核糖体。 然后，我们计划将这些捕获的复合物结晶并解决它们的结构，以填补 我们对这一基本过程的子步骤的了解存在差距。为了连接这些静态， 晶体学衍生的快照核糖体动力学，我们将遵循内部 核糖体的运动使用FRET。我们将利用我们的能力， 重组含有荧光FRET对的活性核糖体， 位置，使我们能够在真实的时间内跟踪核糖体动态， 单分子环境。从这些研究中得出的机制模型可以 通过使用我们现有的方法， 以前开发的靶向突变rRNA或核糖体蛋白，和 在体外测试它们的功能特性。

Project Summary We are entering a new and exciting era of ribosome research, in which the role of ribosome dynamics in protein synthesis is beginning to emerge. This proposal focuses on the role of the structural dynamics of the ribosome, and ribosomal RNA in particular, in the mechanism of translation. Our approach uses a combination of biochemistrty, biophysics, genetics and crystallography. A major goal is to elucidate the mechanism of the coupled translocation of mRNA and tRNA. One strategy is to carry out an exhaustive screen for dominant-lethal mutations in elongation factor EF-G, which we predict will trap the ribosome in previously unobserved transient states of translocation. We then plan to crystallize these trapped complexes and solve their structures, to fill in gaps in our knowledge of the sub-steps of this fundamental process. To link these static, crystallographically-derived snapshots to ribosome dynamics, we will follow internal movements of the ribosome using FRET. We will take advantage of our ability to reconstitute active ribosomes containing fluorescent FRET pairs attached to specific positions, allowing us to follow ribosome dynamics in real time in both ensemble and single-molecule contexts. Mechanistic models derived from these studies can then be tested by creating pure populations of mutant ribosomes using methods that we have previously developed to target mutations to either rRNA or ribosomal proteins, and testing their functional properties in vitro.