The function of an RNA enzyme is intimately related to its tertiary structure. The tertiary structure of RNA arises from formation of defined tertiary interactions. Such interactions lead to proper folding of the RNA, contribute to the specificity of the enzyme-substrate complex and to the transition state of the reaction. The long-term objective of this proposal is to understand the functional and chemical aspects of tertiary interactions in catalytic RNAs. The ribozyme system under study is the catalytic RNA component of an eubacterial ribonuclease P.
The first aim of this proposal is to define the folding domains and to identify nucleotide residues involved in intramolecular tertiary interactions. Folding domains can be localized through constructing bimolecular complexes that combine only through tertiary interactions to restore the catalytic activity. A folding domain is defined when either molecule can fold independently. Active RNase P variants containing alternate tertiary interactions will be selected using a novel in vitro selection procedure. Free energy of these interactions will be determined to elucidate the energetic order of such interactions in defining an RNA structure.
The second aim of this proposal is to determine how this ribozyme recognizes its substrate through tertiary interactions. A series of in vitro selection experiments and site-specific ribose modifications will be performed to pinpoint the nucleotide bases and 2' hydroxyl groups on the substrate and on the ribozyme involved in direct tertiary contacts. Quantitative contributions of these interactions to the ground state and the transition state will be measured to delineate their functional roles.
The third aim of this proposal is to use a unique in vitro selection method to isolate new RNAse P substrates that may or may not resemble the pre-tRNA substrate. These results will deepen our understanding of tertiary structures of RNA, how RNA-RNA recognition occurs through tertiary interactions, and how tertiary interactions influence the transition state in RNA catalysis. Properties of the selected substrates should provide further insights into the mode and versatility of catalysis by the ribozyme from RNase P.
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