How ribonucleic acid (RNA) molecules fold into tertiary structures, how proteins recognize specific RNA structures, and how protein binding is involved in RNA functions, are central questions to a number of biological processes. The broad goal of this proposal is to elucidate structural and mechanistic principles that underlie the formation of a particular RNA structural fold, the loop-loop kissing motif, using multi-dimensional heteronuclear nuclear magnetic resonance (NMR) and fluorescence spectroscopy as the primary tools. RNA loop-loop kissing interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are essential structures involved in a variety of cellular processes in bacteria, in the dimerization of retroviral genomic RNA and in the proper folding of certain messenger and catalytic RNAs. Understanding how RNA kissing interactions function at the molecular level requires detailed studies of the structure, dynamics and protein interactions of these complexes. The proposed studies will focus on two systems in which RNA kissing interactions are observed to nucleate RNA dimerization and specifically direct further RNA refolding through stem strand invasion and helix exchange: the HIV-1 dimerization initiation site (DIS) genomic dimer linkage and the antisense regulation of R1 plasmid repA mRNA translation. The role of the auxiliary proteins HIV-1 nucleocapsid (NCp7) and Gag in chaperoning DIS kissing dimer refolding will also be examined. Specifically, our research will aim to: 1) Examine the role of RNA stem-loop structure in determining the directionality and extent of helix exchange in the repA mRNA-repressor RNA kissing interaction; 2) Examine the role of RNA kissing dimer structure and dynamics in NCp7 chaperoned HIV-1 DIS maturation; 3) Investigate dimerization and maturation of DIS within the context of HIV-1 leader sequences using segmental labeling and fluorescence/NMR spectroscopy; 4) Analyze NCp7 and Gag chaperoned maturation of the DIS kissing dimer and screen for inhibitors of this process. Overall, it is anticipated that these studies will aid in the design of more effective antisense therapies, as well as provide rational approaches for designing inhibitors against HIV-1 DIS, NCp7 and/or Gag that could facilitate development of new antiviral therapeutics.
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