The number of catalytic RNAs and the scope of their functions are expanding at great pace. Yet, our current understanding of RNA catalysis is quite limited. Most mechanistic studies have been concentrated on ribozyme systems that involve phosphodiester bond cleavage or formation. Furthermore, most well studied ribozymes either are cis-active or use oligonucleotide substrates, whose bindings to ribozymes are based on Watson-Crick base-paring. The P.I. has previously isolated a multi-functional RNA, Iso6, that possesses RNA capping, cap-exchange, phosphoryl coupling, pyrophosphatase, and decapping activities. Iso6 catalyzes the formation of a variety of molecules containing "high energy" phosphoanhydride bonds from natural and biologically important terminal phosphate-containing molecules. This class of "High energy" phosphoanhydride-bridged molecules, such as mRNA caps, CoA, NAD, FAD, NDP-sugars, and signaling molecules A(5 cents)pppA, A(5 cents)ppppA, etc., are widespread in the cell and play critical roles in cellular processes. In addition, several engineered versions of Iso6 recognize multiple small-molecule substrates whose binding does not rely on conventional Watson-Crick base-paring, and catalyze the formation of larger products with multiple turnovers. This project aims to probe and define the structure around the catalytic center and to investigate the reaction intermediates of Iso6. Phosphorothioate substitutions will be introduced into Iso6 around its reaction center, as well as into the RNA's substrates to locate the required calcium ion. Different probes will be linked to substrates to map the two substrate sites within the RNA. Detection, isolation, and characterization of potential reaction intermediates will be undertaken to define the reaction mechanism of Iso6. The studies of Iso6 will help understand the new type of RNA-catalyzed chemistry of "high energy" phosphoanhydride bond formation, and may provide evidence to suggest that the broad spectrum of biologically important phosphoanhydride-bridged molecules might have arisen in an RNA world. Investigations of Iso6's reaction center will address a fundamental question of RNA catalysis: how is RNA able to bind and juxtapose multiple small-molecule substrates so that reactions occur rapidly? Successful isolation of new reaction intermediates from Iso6 may establish new reaction pathways of RNA catalysis. Information obtained throughout this research will advance our understanding of RNA catalysis in general.
2. Non-technical
Catalytic RNA molecules, or ribozymes, play important roles in the cell. With the recent development of in vitro molecular evolution techniques, the number of new catalytic RNAs and the scope of their functions are expanding at great pace. However, comparing our knowledge of protein enzyme catalysis, our current understanding of RNA catalysts is quite limited. Most mechanistic studies on ribozymes have been concentrated on ribozyme systems that involve phosphodiester bond cleavage or formation. Furthermore, well studied ribozymes either are cis-active or use oligonucleotide substrates, whose binding to ribozymes is based on Watson-Crick base-paring. Accordingly, it is highly desirable to study an RNA enzyme mechanism based on ribozyme systems that function like proteins. The objective of this study is to probe and define the structure and reaction mechanism of a multifunctional RNA enzyme, Iso6, which was previously isolated by the principle investigator. Iso6 catalyzes the formation of a variety of molecules containing "high energy" phosphoanhydride bonds from natural and biologically important terminal phosphate-containing molecules. Phosphorothioate substitutions and different probes will be used to map the reaction center within the RNA. Detection, isolation, and characterization of reaction intermediates will be undertaken to define the reaction mechanism. The results will help to understand the new type of RNA-catalyzed chemistry of "high energy" phosphoanhydride bond formation, to suggest new reaction pathways of RNA catalysis, and to address a fundamental question of RNA catalysis: how is RNA able to bind and juxtapose multiple small-molecule substrates so that reactions occur rapidly?
