This proposal describes our systematic approach directed toward understanding the relationships between structure, stability and function in a family of structurally related RNA pseudoknots. Pseudoknots are an element of RNA structure that are widespread in nature, and play an integral role in regulating a variety of biological functions. The research plan will focus on RNA pseudoknots within the autoregulatory gene 32 mRNAs of bacteriophages T2, T4 and T6, and the frameshift- regulating pseudoknot within the gag-pol mRNA of the SRV lentivirus (simian AIDS). Preliminary evidence strongly suggests that the bacteriophage and the SRV pseudoknots are representative members of a single large family of structurally related RNA pseudoknots, the members of which differ in function. Specifically the following experiments are proposed: (1) The structures of the bacteriophage T2 gene 32 autoregulatory pseudoknot and the simian retrovirus (SRV) gag-pol frameshift directing pseudoknot will be determined at high resolution using multidimensional NMR methods. Since the functions of the pseudoknots are divergent, a comparison of their structures will provide insight as to which of the common features of the pseudoknot family are conserved for functional versus structural reasons. (2) The RNA pseudoknots of the bacteriophage T4 and T2/T6 gene 32 non-coding regions will be used as model systems to gain an understanding of the stability of the structually deemed paradigm of this class of molecules. A set of systematic base changes will be introduced into the terminal and internal regions of the T4-encoded pseudoknot and determine how the physical properties are perturbed relative to the wild-type RNA. Physical properties will be measured using ultraviolet absorbance melting, chemical and ribonuclease cleavage and imino proton NMR measurements collected as a function of temperature and solution conditions. Nucleotide changes will be guided by an extensive nearest-neighbor thermodynamic database derived from model oligoribonucleotides from which predictions of the effect of nucleotide changes are possible.
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