INTERACTION OF RIBOSOMAL S15 PROTEIN WITH RRNA

本项目的总体目标是了解 特异性相互作用S15和16 SrRNA。 我们将开始定义 使用组分形成复合物所需的最小蛋白质和RNA 来自B。嗜热脂肪菌 我们将描述自由的结构 蛋白质、游离RNA和S15-rRNA复合物的NMR光谱，以及 荧光能量转移 我们希望了解16 S RNA 结构有助于S15的识别，以及S15的结合是如何进行的。 组织核糖体核糖核酸，使核糖体组装可以进行。 具体 目标如下： 1. 确定S15蛋白的最小rRNA结合位点。 我们将 开始通过鉴定最小的16 S RNA片段，完全包含 S15的结合位点。 2. S15蛋白和蛋白片段的结构研究。 我们将 作为必要步骤，使用NMR光谱法表征游离蛋白质 用于研究RNA-蛋白质复合物。 3. 制备设计用于大RNA NMR的氘代核糖核苷酸。 在 为了便于大RNA和RNA-蛋白质的NMR研究 复合物，我们将制备核糖核苷酸，其中3 '，4'，5 '和5” 蛋白质已经被氘核所取代。 4. 游离最小rRNA片段的结构研究。 我们将研究 最小rRNA结合位点的结构使用NMR光谱。 5. S15结合后rRNA构象变化的表征 使用荧光能量转移。 S15的结合可能伴随着 rRNA的构象变化 我们将构建RNA片段 由多条RNA链组成，这些RNA链在 3 '末端以估计蛋白质之前和之后的末端到末端距离 约束力 6. 最小S15-rRNA复合物的结构研究。 我们将 表征最小S15-最小16 S RNA复合物的结构 使用多维核磁共振光谱。 我们感兴趣的是 结构有助于结合，RNA-蛋白质的性质 接触，以及蛋白质诱导的构象变化的性质。 7. 表征S6和S18的后续结合，作为 核糖体装配 从S15-rRNA复合物开始，我们将确定 最小的rRNA片段能够结合这三种蛋白质， 由S6/S18形成的额外相互作用。

The overall goal of this project is to understand the structural basis for specific interaction S15 and 16S rRNA. We will begin by defining the minimal protein and RNA requirements for complex formation using components from B. stearothermophilus. We will characterize the structure of the free protein, free RNA, and of the S15-rRNA complex using NMR spectroscopy, and fluorescence energy transfer. We hope to learn about how the 16S RNA structure contributes to the recognition of S15, and how the binding of S15 organizes the rRNA such that ribosomal assembly can proceed. The specific Aims are as follows: 1. Determine the minimal rRNA binding site for the S15 protein. We will begin by identifying the minimal 16S RNA fragment that completely contains the binding site for S15. 2. Structural studies of the S15 protein and protein fragments. We will characterize the free protein using NMR spectroscopy as a necessary step for studying the RNA-protein complex. 3. Prepare deuterated ribonucleotides designed for NMR of large RNAs. In order to facilitate the NMR studies of large RNAs and RNA-protein complexes, we will prepare ribonucleotides where the 3', 4', 5', and 5" proteins have been replaced by deuterons. 4. Structural studies of the free minimal rRNA fragment. We will study the structure of the minimal rRNA binding site using NMR spectroscopy. 5. Characterization of the rRNA conformational change upon S15 binding using fluorescence energy transfer. Binding of S15 may be accompanied by a conformational change in the rRNA. We will construct RNA fragments composed of multiple RNA strands that are fluorescently labeled at the 3'ends to estimate the end to end distances before and after protein binding. 6. Structural studies of the minimal S15-rRNA complex. We will characterize the structure of the minimal S15- minimal 16S RNA complex using multidimensional NMR spectroscopy. We are interested in how the RNA structure contributes to the binding, the nature of the RNA-protein contacts, and the nature of the protein-induced conformational change. 7. Characterize the subsequent binding of S6 and S18, as a model for ribosomal assembly. Beginning with the S15-rRNA complex, we will determine the minimal rRNA fragment competent to bind these three proteins, focusing on the additional interactions formed by S6/S18.