Structural and Functional Investigations of Conformationally Interconverting RNA Pseudoknots
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The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.
RNA structure and function
Stammler, Suzanne (2009). Structural and Functional Investigations of Conformationally Interconverting RNA Pseudoknots. Doctoral dissertation, Texas A&M University. Available electronically from