The long term goal of this project has been to explore molecular interactions in RNA-protein complexes from the ribosome and relate those properties to the functional role of the complex within the ribosome. The results from these studies are anticipated to define, in structural and thermodynamic terms, novel aspects of RNA tertiary structure, the ways in which ligands recognize and manipulate an RNA structure, and the relationship between these interactions and the function of the protein-RNA complex as part of the ribosome. The present work will focus on the interactions of a highly conserved, 58 nucleotide domain of the large subunit ribosomal RNA with a conserved protein, L11, and antibiotics; in particular, a hypothesis that the protein binds rRNA in two different modes, both relevant to ribosome function, will be tested. Three kinds of work will be carried out: (1) Structural studies of different protein fragment- and antibiotic-RNA complexes, using """"""""footprint"""""""" studies of both protein and RNA, as fragments and in situ in the ribosome, and also using NMR to define the protein and RNA structures and ligand - RNA contact surfaces. (2) Thermodynamic description of L11, L11 fragment, and antibiotic binding to rRNA, including the specific uptake of Mg2+ and NH4+ ions. Calorimetry and gel assays will be used to measure binding affinities and associated enthalpies and heat capacity changes. (3) Functional studies of L11 and antibiotics in the intact ribosome, starting with the effects of these ligands on elongation factor-G dependent GTPase activity. Results from these studies should contribute to a basic understanding of protein - RNA recognition mechanisms, as well as elucidation of the structure and function of a highly conserved region of the ribosome.
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