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Chan, Philip A.

Microbiology and Molecular Genetics

2004

M.S.

Fluorescence techniques are commonly applied to provide real-time data on dynamic changes in the structure of various protein and nucleic acid models. The hairpin ribozyme is a small catalytic RNA which has been studied using fluorescence molecules to report global as well as local alteration in structure. In the present work, the fluorescent probe 2-aminopurine (2-AP) has been used to gather data on specific changes during folding and catalysis of the hairpin ribozyme. 2-AP is used to study the fluorescence quenching accompanying native folding of the RNA at site U+2 in the substrate, and nucleotides G8 and A38 in the ribozyme. The 2-AP substitution at U+2 shows substantial unstacking yet restricted rotational movement in the active structure, demonstrating an important conformational change proceeding catalysis. Similarities were noted between protein folding studies and a possible kinetic trap formed as a result of U+2 mutations. Fluorescence quenching assays were further used to take advantage of the pH -dependent nature of the 2-AP Nl position.
Substitution of 2-AP at G8 in the ribozyme revealed an Nl pKa shifted toward neutrality, supporting a possible model in which G8 is directly involved in acid-base catalysis of the substrate. Irradiation of 2-AP with UV light causes interstrand crosslinking of the 2-AP and forms identifiable products with bases upon which it stacks. Using this method, the potentially catalytic base A38 was found to crosslink to G+1, the base directly adjacent to the scissile bond. The purified 2AP38/G+1 crosslink retains cleavage activity and presumably represents a step during or immediately proceeding cleavage. The crosslink positions A38 in the correct orientation as a direct participant in the cleavage reaction, possibly as the partner to G8 in acid-base catalysis.