Chemical catalysis in ribozymes

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The discovery of catalytically active ribonucleic acids, known as ribozymes, redefined the role of RNA in biology. Despite the recent progress in RNA structure determination, the detailed mechanisms by which RNA mediated catalysis is achieved remain elusive. In comparison to protein enzymes, RNA utilizes only four different building blocks and has a much more limited set of functional groups available. Thus the question arises of how ribozymes can achieve their impressive catalytic acceleration of reaction rates. Recent results from our laboratory suggest that the internal electrostatic field within the ribozyme active sites significantly contributes to the catalytic potential of RNA. We will utilize two RNA systems for the proposed studies, the natural Varkud satelite ribozyme, and the in vitro selected malachite green binding RNA aptamer, which we engineered in to a primitive catalyst. We will study the structure of the VS ribozyme active site domain in the context of the complete ribozyme and characterize chemical reactions of malachite green derivatives inside and outside the RNA binding pocket using NMR spectroscopy, Ultraviolet/Visible spectroscopy, as well as computational approaches. Furthermore we will characterize the forces and intramolecular interactions that enable these ribozymes to catalyze chemical reactions. The long-term objectives of the work described in this grant are to understand how molecular structure and forces within these ribozymes contribute to catalysis. Specifically we are asking: What are the structures of the RNA catalytic centers? How do these structures affect the charge distribution and chemical properties of nucleotides that are involved in catalysis? How important are electrostatic forces in RNA catalysis? And can we draw general mechanistic conclusions about biological catalysts from the chemically relatively simple ribozymes? The studies will also help to lay the groundwork for a systematic design of new ribozymes with altered specificities that can be used as tools in molecular biology, chemistry, nanotechnology, and medicine.