Poster abstracts
Poster number 162 submitted by Neil White
The relationship between conformational dynamics and catalysis in the lead-dependent ribozyme
Neil A. White (Department of Biochemistry and Molecular Biology, Michigan State University), Minako Sumita (Department of Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Department of Biochemistry and Molecular Biology, Michigan State University)
Abstract:
In both NMR and crystallographic structures of the lead-dependent ribozyme, or leadzyme, a conformational rearrangement is necessary to achieve the in-line geometry appropriate for self-cleavage. Thus, we and others have hypothesized that there is a transition from the inactive, ground-state structure to an active conformation which exists a small percentage of the time. Testing such models requires analyzing which molecular motions exist and also, whether the detected motions are catalytically relevant. In other words, we study dynamics-function relationships, in analogy with the classic structure-function relationship. In this work, we report NMR spin relaxation studies in combination with metabolically-driven specific stable isotope labeling to detect microsecond scale motions of the ribose group at the active site of the leadzyme. To test the functional relevance of this apparent ribose repuckering mode, we supplement our previously-reported application of locked nucleic acid (LNA) nucleotides with a modified structure that retains the nucleophilic 2′-hydroxyl while covalently restricting the ribose conformation to C3′-endo. The combined results are consistent with a dynamic repuckering of the ribose group adjacent to the scissile phosphate being a critical step in the ribozyme catalytic mechanism.
Keywords: leadzyme, conformational dynamics, RNA catalysis