Ribonucleic acids (RNAs) and RNA folding motifs play central roles in the structure and assembly of ribonucleoprotein particles, as well as regulatory roles in translation and gene silencing through RNA interference. An RNA pseudoknot is a simple RNA folding motif composed of two helical stems connected by two single-stranded loops. Pseudoknots play prominent, though poorly understood, roles in translation initiation and in mRNA receding, including stop codon suppression and ribosomal -1 frameshifting. Our long term goal is to elucidate and ultimately manipulate, through drug design, how RNA structure, stability, and rates of ribosomal pausing and pseudoknot unfolding during the translation elongation cycle dictate the ability of RNA pseudoknots to stimulate -1 mRNA frameshifting in plant and animal RNA viruses.
Our specific aims are to: 1) Refine our solution structure of the P1-P2 mRNA pseudoknot encoded by the luteovirus, pea enation mosaic virus RNA-1 (PEMV-1); 2) Solve the high resolution solution structure of the P1 -P2 mRNA pseudoknot encoded by sugarcane yellow leaf virus (ScYLV) as a means to further elucidate the general rules that govern formation of intramolecular triple helical structures in RNA; 3) Solve the high resolution solution structure of the HIV-1 gag-pol frameshifting pseudoknot, which recent functional studies suggest contains a minor groove intramolecular triplex, as well as a potential major groove-derived protonated C+.(G-C) triple base pair, both features common to all plant luteoviral pseudoknots; 4) Use the luteoviral P1-P2 mRNA system to evolve tight-binding peptides using an in vitro selection strategy based on mRNA display; and 5) Develop novel single-molecule fluorescence-based assays to examine the real-time kinetics of ribosomal pausing and pseudoknot unfolding of functional vs. nonfunctional MMTV gag-pro pseudoknots during translation elongation, as a test of the torsional restraint model of pseudoknot-mediated frameshift stimulation. These experiments will significantly enhance our understanding of how mRNA structure, stability and folding influence ribosomal recoding required for replication and propagation of infectious plant and human RNA viruses.
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