Investigation of metal-ion binding in the four-way junction construct of the hairpin ribozyme

摘要

The hairpin ribozyme is a small catalytic RNA that cleaves a phosphodiesterbond. In order for cleavage to occur, the hairpin ribozyme must properly fold into itsdocked conformation, in which the two loops interact to form the active site. Metal ionsand the four-way junction play critical roles in the stabilization of the dockedconformation. The work presented in this thesis attempts to investigate the metal-iondependence of the docking of the four-way junction construct of the hairpin ribozyme. Inaddition, the activity of the hairpin ribozyme in the presence of Mn2+ was observed.Initially, a four-stranded four-way junction construct of the hairpin ribozyme and aloopless mutant were characterized by native gel electrophoresis and thermaldenaturation to verify ribozyme formation.A novel interaction between the sulfur of a phosphorothioate-substitutedmononucleotide, such as adenosine thiomonophosphate (AMPS) or adenosinethiotrisphoshate (ATPgS), and Cd2+ has been characterized by UV-vis spectroscopy. Afeature at 208 nm was identified to be a result of sulfur-to-Cd2+ transfer. The apparentbinding affinities, the apparent extinction coefficients, and the binding ratios weredetermined for each complex.