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RNAs and RNA-protein complexes, we will prepare ribonucleotides where the 3', 4', 5', and 5"""""""" proteins have been replaced by deuterons. 4.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