The broad goal of this research program is to understand fundamental aspects of RNA structure and catalytic function. The RNA subunit of bacterial ribonuclease P (Rnase P), which catalyzes the specific cleavage of pre-tRNA, has been chosen for study because it represents an essential, widespread and conserved call of catalytic RNA molecules (ribozymes). The specific elements of the research program are: 1. Two complementary approaches will be employed to pinpoint the precise residues on the ribozyme involved in substrate-binding and catalysis. Intermolecular crosslinking, using both randomly and site-specifically modified substrates, will be used to determine riboyme nucleotides juxtaposed to known contact sites on the substrate. Residues where chemical modification interferes with binding or catalysis will be identified by employing a series of novel nucleotide analogs in modification-interference experiments. These results are expected to suggest specific elements of structure, including intermolecular contacts. 2. The reaction kinetics of ribozymes containing site- specific functional group modifications will be examined to test potential interactions indicated by the analyses of Specific Aim 1. Steady-state, single-turnover and binding kinetics will be measured in order to differentiate between effects on catalysis and binding. A novel strategy employing self-cleaving ribozyme-substrate conjugates will be used to simplify determination of catalytic rate of mutant or modified ribozymes. 3. Tertiary contacts within the ribozyme will be determined by analyzing the association of isolated Rnase P RNA domains in vitro. To facilitate detection of binding, pre-tRNA sequences will be fused to individual domains and inter-domain interactions will be assayed by intermolecular cleavage. 4. The dynamics of ribozyme structure including folding and substrate-induced conformational changes will be analyzed using a photoaffinity crosslinking approach. Analysis of folding will reveal the order of formation of secondary and tertiary interactions and will include an assessment of the chemical factors which influence their formation. Identification of structural differences between the free ribozyme and ribozyme-substrate complex will help define rearrangements that accompany the multiple turnover RNase P reaction. The data provided by the research program will shed light on fundamental aspects of RNA function; including structure, the nature of RNA-RNA interactions, and RNA-mediated catalysis. A better comprehension of these issues will lead to improvement in our understanding of the guiding principals for engineering RNA-based therapeutics which offer enormous potential for new directions in treatment of disease.
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