Ribosome Structure and Function

 描述（由申请人提供）：我们正在进入一个新的和令人兴奋的核糖体研究时代，其中核糖体动力学在蛋白质合成中的作用开始显现。这个建议的重点是核糖体的结构动力学的作用，特别是核糖体RNA，在翻译机制。我们的方法结合了生物化学、生物物理学、遗传学和晶体学。一个主要的目标是阐明mRNA和tRNA的耦合易位的机制。一种策略是进行一个详尽的屏幕上的延伸因子EF-G，我们预测将陷阱的核糖体在以前未观察到的瞬时易位的显性致死突变。然后，我们计划结晶这些被困的复合物，并解决他们的结构，以填补我们的知识空白， 这一基本过程的子步骤。为了将这些静态的、晶体学衍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。我们将利用我们的能力，以重建含有荧光FRET对连接到特定位置的活性核糖体，使我们能够遵循核糖体动力学在真实的时间在合奏和单分子上下文中。然后，可以通过使用我们以前开发的将突变靶向rRNA或核糖体蛋白的方法创建纯的突变核糖体群体，并在体外测试其功能特性，来测试来自这些研究的机制模型。

 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.