Ribosomal RNA is no longer considered a purely structural component of ribosomes, but thought perhaps to play the central catalytic role in protein synthesis. This reassessment of its role has been prompted in part by methodological advances in studies of RNA structure-function correlations, and also in part by the discoveries of other catalytically active RNA molecules, such as RNase P. How """"""""simple"""""""" macromolecules like RNA, consisting only of four monomers, can function catalytically is a significant chemical-biological question. A key feature of such RNA sequences is thought to involve conformational flexibility. A more thorough understanding of the structural basis of conformational flexibility in nucleic acids will inform all areas of molecular biology and, by extension, those areas of medical science upon which molecular sciences directly impinge. In the proposed project, a specific hypothesis regarding the functional importance of conformational flexibility in the HelixII-HelixIII region of 5S ribosomal RNA will be tested. The experimental approach employs a combination of genetic, biochemical, and physical-chemical methods to answer the following questions: (1) is the potential for conformational flexibility in 5S RNA biologically significant? (2) Is 5S RNA in fact conformationally flexible in the sense that the HelixII-III region can switch between two (or more) secondary structures in solution? (3) If switching occurs, what are the energetics and dynamics of the conformational change and what are the three- dimensional structures of the states involved? Functional studies aimed at answering Question (1) will be carried out first to demonstrate the biological significance of the project, before proceeding with questions (2) and (3), which involve a greater commitment of resources, in particular the intensive use of high- field NMR spectroscopy. Mutations will be constructed in a cloned gene coding for the eubacterial 5S RNA from E.coli, which will alter the RNA's primary structure in ways that are expected to alter its conformational flexibility. Altered 5S RNA's will be prepared by in vitro or in vivo transcription of the mutated genes and tested for biological function, including binding to ribosomal proteins L25 and L18, and incorporation into 50S ribosomal subunits. Successfully reconstituted ribosomes containing the altered RNAs will be tested for tRNA binding and protein synthesis. High-field, two-dimensional NMR techniques will be undertaken to characterize the solution structure(s) and conformational dynamics of the molecule. The proposed structural studies will be carried out on (1) short RNA sequences which are designed to model the two-base bulge structure in Helix III (2) RNA sequences which model the entire switch region of Helices II and III (normal sequence as well as altered, conformationally """"""""locked"""""""" sequences); and (3) the intact 5S RNA molecules bearing these same mutations.
