This proposal requests continued funding for ongoing studies of RNA pseudoknots, directed toward understanding the relationships between structure, stability, dynamics and function within this important and widespread RNA motif. The research plan focuses on the pseudoknots from the autoregulatory gene 32 mRNAs of T-even bacteriophages, and the frameshift-stimulating pseudoknots from the pathogenic retroviral gag-pro mRNA mu1 frameshift sites. The following experiments are proposed: 1) Multi-dimensional NMR spectroscopy will be used to determine the solution structure of the gag-pro frameshifting pseudoknot of mouse intracisternal A-particle (mIAP), complementing the applicant's recently published NMR investigations of the pseudoknot at the simian retrovirus gag-pro frameshift site; 2) Thermodynamics of folding of the bacteriophage and retroviral pseudoknots will be investigated using optical melting techniques and differential scanning calorimetry to define stability determinants; 3, 4) The conformational dynamics and kinetics of pseudoknot unfolding will be investigated using NMR-based and steady-state fluorescence spectroscopies. Results will indicate whether conformational switching occurs on a time scale which is compatible with translational elongation and therefore ribosomal frameshifting; 5) Fluorescence resonance energy transfer (FRET) spectroscopy will be used to compare the global flexibilities of the bacteriophage gene 32, MMTV and mIAP pseudoknots, as well as define the distribution of end-to-end distances compatible with the pseudoknot structures, thus providing a direct comparison of """"""""straight"""""""" pseudoknots with coaxial stems and the """"""""bent"""""""" pseudoknots that stimulate frameshifting at the retroviral gag-pro interface. In the case of the retroviral pseudoknots, results will provide insight into the requirements for stimulating a frameshift upon translation of mRNA by the ribosome, and may ultimately provide insight as to how to target the pseudoknot in order to interfere with its essential function.
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