Ribosome Structure and Function

 描述(申请人提供)：我们正在进入一个新的和令人兴奋的核糖体研究时代，在这个时代，核糖体动力学在蛋白质合成中的作用开始显现。这一建议侧重于核糖体的结构动力学，特别是核糖体RNA在翻译机制中的作用。我们的方法结合了生物化学、生物物理学、遗传学和结晶学。一个主要的目标是阐明mRNA和tRNA偶联易位的机制。一种策略是对延伸因子EF-G的显性致死突变进行详尽的筛查，我们预测这将使核糖体陷入以前未观察到的瞬时易位状态。然后我们计划将这些被捕获的络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。为了将这些静态的、结晶学派生的快照与核糖体动力学联系起来，我们将使用FRET跟踪核糖体的内部运动。我们将利用我们重建活性核糖体的能力，这些核糖体含有连接到特定位置的荧光FRET对，使我们能够在整体和单分子环境中实时跟踪核糖体的动态。然后，可以通过使用我们之前开发的针对rRNA或核糖体蛋白质的突变的方法创建突变核糖体的纯种群，并在体外测试它们的功能特性，来测试从这些研究中获得的机制模型。

 DESCRIPTION (provided by applicant): 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 biochemistry, 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.