What is the function of distinct nucleobases in the catalytic mechanism of the hairpin ribozyme?

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RNA catalysis plays an important role in processing and translation of ribonucleic acids but the catalytic mechanism remains unclear in most cases. The hairpin ribozyme catalyzes cleavage of RNA releasing a fragment with a free 5-OH group and one with a 2-3-cyclic phosphate. This research proposal addresses the question if the catalytic mechanism of the hairpin ribozyme is based on general acid-base catalysis by the distinct nucleobases A38 (acid) and G8 (base) or if these purine bases only provide electrostatic stabilization of the transition state. Different approaches will be used to address this question. In all cases, hairpin ribozymes will be assembled from chemically synthesized RNA molecules. First of all, a substrate will be synthesized in which the 5-oxygen of the scissile phosphate is replaced by sulfur (5-PS). This highly activated substrate will not require general acid catalysis for its cleavage and replacement of the acid A38 by other nucleobases should not inhibit the catalytic function. Secondly, the 5-PS substrate will allow to investigate the pH profile of the reaction independently from the acid. Then, ribozymes with modified nucleobases of lower pKa at the G8 position could be used to proof the role of G8 as general base. A third approach will be to concentrate on the transition state of the reaction. For this, kinetic isotope effects of reacting atoms will be investigated. In summary, using a combination of chemistry and the methods of mechanistic enzymology, it should be possible to elucidate the catalytic mechanism of the hairpin ribozyme. Understanding the catalytic mechanisms of different ribozymes is important for several reasons. First, they perform reactions necessary for the metabolism in living cells. Second, they can potentially provide a glimpse into the evolution of life and the possible existence of an RNA world. Lastly, as simplified macromolecular catalysts they provide mechanistic insight into biocatalysis in general.