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Investigation of metal binding properties in the hairpin ribozyme
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The hairpin ribozyme is a small catalytic RNA originally found in the negative strand of the tobacco ringspot virus. It possesses a unique four-arm RNA structure containing four helices (helices 1-4) and five loops (loops 1-5), which form two independently folding domains, labeled loop A and loop B. Divalent metal ions are thought to bind in specific sites within the structure and promote ribozyme folding, substrate binding, and cleavage. The goals of the present studies are to further clarify the metal ion interactions that are required by the hairpin ribozyme for both structural and catalytic purposes. Thermal denaturation studies were carried out in an effort to elucidate unfolding pathways and study the Mg²⁺ dependence of these unfolding events. Examination of the melting profile indicates that the hairpin ribozyme unfolds in three major transitions. In the full hairpin the two loops unfold independently with loop A unfolding before loop B. No transitions have been correlated to unfolding of tertiary interactions between the loops. Results were confirmed with a hairpin construct known to lack interloop contacts. Hairpin activity has been characterized by measuring the cleavage rates and dependence on both Mg²⁺ and Mn²⁺ under several different buffer conditions. Apparent affinities for sites relevant to catalysis are ~ 4 mM for Mn²⁺ and ~ 10 mM for Mg²⁺ in 0.1 M NaCl. Biphasic rate behavior was observed, suggesting multiple conformations of hairpin ribozyme. Rates were performed in the presence of urea in an attempt to stabilize the correctly folded hairpin RNA. Rates of cleavage showed a maximum 1 M urea. Mg²⁺ dependence was not affected by addition of urea. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain a direct measurement of the numbers and affinities of Mn²⁺ ions that bind to the hairpin ribozyme and the separated loop A and loop B constructs. These affinities (K = 50 M in 0.1 M NaCl) all appear to be higher than predicted for purely nonspecific interactions (K ~ 147 M) (Vogt, unpublished). The number of Mn²⁺ bound in both loop A-d14 and loop B are each approximately half those found for the complete hairpin ribozyme, suggesting few new sites are created by hairpin folding.
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Includes bibliographical references (leaves 96-112).
Issued also on microfiche from Lange Micrographics.
Kirchner, Alyson Jane (2002). Investigation of metal binding properties in the hairpin ribozyme. Master's thesis, Texas A&M University. Available electronically from
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