INTERACTION OF RIBOSOMAL S15 PROTEIN WITH RRNA

核糖体 S15 蛋白与 RRNA 的相互作用

• 批准号: 2332020
• 负责人: James R Williamson
• 金额: $ 17.19万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332020
• 关键词: Bacillus stearothermophilus; chemical binding; computer simulation; conformation; deuterium; fluorescence spectrometry; fluorescent dye /probe; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid structure; physical model; protein structure function; ribosomal RNA; ribosomal proteins; stoichiometry; structural biology; synthetic nucleotide

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of large RNAs and RNA-protein complexes, we will prepare ribonucleotides where the 3', 4', 5', and 5" proteins have been replaced by deuterons. 4.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.

该项目的总体目标是了解 特定相互作用S15和16S rRNA。 我们将首先定义 使用组件的最小蛋白质和RNA的需求 来自B. stearothothrophilus。 我们将表征自由的结构 使用NMR光谱法，蛋白质，游离RNA和S15-RRNA复合物的蛋白质，并 荧光能量转移。 我们希望了解16S RNA如何 结构有助于识别S15，以及S15的结合如何 组织rRNA，使核糖体大会可以进行。 具体 目的如下： 1。确定S15蛋白的最小rRNA结合位点。 我们将 首先确定完全包含的最小16S RNA片段 S15的结合位点。 2。S15蛋白质和蛋白质片段的结构研究。 我们将 使用NMR光谱法表征自由蛋白作为必要的步骤 用于研究RNA蛋白质复合物。 3。准备为大型RNA的NMR设计的氘化核糖核苷酸。 在 为了促进大型RNA和RNA-蛋白的NMR研究 复合物，我们将准备3'，4'，5'和5英寸的核糖核苷酸 蛋白质已被杜特龙取代。 4。自由最小rRNA片段的结构研究。 我们将学习 使用NMR光谱法最小rRNA结合位点的结构。 5。rRNA构象变化在S15结合上的表征 使用荧光能量转移。 S15的结合可能伴随 rRNA的构象变化。 我们将构造RNA片段 由多个RNA链组成，这些链被荧光标记在 3'端以估计蛋白质之前和之后的端到端距离 结合。 6。最小S15-RRNA复合物的结构研究。 我们将 表征最小S15-最小16S RNA复合物的结构 使用多维NMR光谱法。 我们对RNA感兴趣 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。将S6和S18的随后结合表征为模型 核糖体组件。 从S15-RRNA复合物开始，我们将确定 最小的rRNA片段有能力结合这三种蛋白质，聚焦 在由S6/S18形成的其他相互作用上。