Poster abstracts
Poster number 137 submitted by Neil White
Ribose Dynamics of the Leadzyme
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:
The lead-dependent ribozyme or leadzyme is among the smallest of the known catalytic RNAs. Lead-dependent cleavage occurs between C6 and G7 within a six-nucleotide asymmetric internal loop. In structures of the leadzyme determined by NMR spectroscopy and X-ray crystallography the scissile phosphate is not positioned for in-line nucleophilic attack. This necessitates a conformational rearrangement of the active site to be consistent with the proposed transition state. We have previously probed the ribose structure and dynamics of the guanosine residues in the asymmetric loop using LNA (locked nucleic acid). We now report a study of the relationship between conformational dynamics and catalytic function at the cleavage-site residue C6. We have substituted C6 with a 2’-hydroxyl-bicyclo [3.1.0] hexane nucleotide, which is restricted to the C3’-endo ribose conformation but, unlike LNA, maintains the nucleophilic 2’-hydroxyl, and found a drastic attenuation of self-cleavage activity. In parallel, we have undertaken NMR spin relaxation experiments to examine ribose repuckering events at the C6 position using our previously-reported site-specific C-13 isotopic labeling scheme. These studies in totality will yield improved understanding of the relationship between RNA backbone dynamics and cleavage mechanism in the leadzyme.
Keywords: leadzyme, conformational dynamics, RNA catalysis