This proposal consists of three projects which all involve studying the structure of isolated domains of larger RNA molecules and understanding how that structure is related to biological function. Recently developed methods for the in vitro transcription of synthetic DNA with T7 RNA polymerase will be refined and used to prepare RNA fragments of defined sequence and approximately 20 nucleotides in length. The structure of these fragments and carefully designed sequence variants will be studied using well established chemical modification methods and by nuclear magnetic resonance techniques. Attempts will be made to prepare diffraction quality crystals. A study of the interaction of bacteriophage R17 coat protein with the replicase translational operator region of R17 RNA will be continued with the focus on the protein. A combined biochemical and molecular biological approach will be used to identify amino acids involved in RNA binding and protein-protein association. The role of this interaction in translational repression and phage assembly will be investigated using both in vitro and in vivo experiments. Recent experiments demonstrating a new, very small RNA enzyme will be extended. By constructing variants of the active sequences, both the structure and the mechanism of the reaction will be studied.
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