RNA PSEUDOKNOT STRUCTURE AND STABILITY

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My long-term career goal is to maintain a strong research program in an area of fundamental biomedical importance. In the first few years of my career as an independent scientist, I believe I have already made a substantial contribution to our understanding of RNA structure, the structural basis of RNA-protein recognition and binding, RNA pseudoknots, and components of the translational apparatus associated with ribosomal frameshifting and re-coding. The salary support provided by an Independent Scientist Award (K02) will relieve me of half of my present teaching load (currently approximately 80 lectures per year), and provide relief from administrative duties, freeing 75 to 80 percent of my time for research. The additional research time made available by this Independent Scientist Award will have an immediate and positive effect on my NIH-funded research productivity. The present proposal is based on my recently funded NIH (R01) proposal, directed toward understanding the relationships between structure, stability and function in a family of related RNA pseudoknots. Pseudoknots are an element of RNA structure that is widespread in nature, playing an integral role in a variety of biological processes. Investigations will focus on RNA pseudoknots from the gene 32 mRNA of bacteriophage T2, and the gag-pro mRNA frameshift site of the SRV-1 lentivirus. Preliminary evidence strongly suggests that the bacteriophage and these pseudoknots are representative members of a single large family of related RNA pseudoknots, the members of which differ in function, but share a common structural motif. The following experiments are proposed: (1) The detailed structures of the bacteriophage T2 and SRV-1 pseudoknots will be determined using multi- dimensional NMR methods. A comparison of the pseudoknot structures will provide insight as to which of the common features of the pseudoknot family are conserved for functional versus structural reasons. (2) Previous results suggest that the SRV-1 frameshift signal may also be embedded within the pseudoknot stability, dynamics and unfolding pathway, as well as the structure. A series of NMR experiments will be used to investigate in detail these dynamic aspects of the RNA pseudoknots.