This proposal will continue structure-function studies on the hammerhead ribozyme. The hammerhead ribo/yme has been shown to efficiently cleave specific RNA targets both in vitro and in vivo. Thus these ribozymes have potential biomedical applications for inactivation of specific gene functions by cleavage of mRNA or as antiviral agents by cleavage of the genome of RNA viruses. The work in this proposal focuses on detailed studies of the structure and dynamics of the hammerhead ribozyme, primarily by multi- dimensional heteronuclear nuclear magnetic resonance (NMR) techniques. Recent studies have shown that a slightly larger domain of naturally occurring plant viroid hammerhead ribozymes has a much faster cleavage rate (-100 fold faster) than the previously characterized hammerhead systems. Thus the main focus of this proposal will be probing the structure and dynamics of these faster hammerhead ribozymes. Isotopically '3C/'5N-labeled ribozymes will be prepared by in vitro transcription with T7 RNA polymerase and a variety of multi-dimensional heteronuclear NMR experiments will be performed to make resonance assignmentand obtain structural information on the hammerhead ribozyme. Nuclear Overhauser effect, J-couplingconstants and residual dipolar couplings data will be collected on various hammerhead constructs and used to determine the local and global structure of these ribozymes in solution. Heteronuclear 13C NMR relaxationexperiments will be performed to measure dynamics on these ribozymes. These relaxation experiments give information on the conformational dynamics of individual residues in the RNA, and probe exchange processes with lifetimes from micro- to milliseconds. Both ensemble and single molecule fluorescence resonance energy transfer techniques will be used to study tertiary interactions and dynamics for the hammerhead. One of the intriguing properties of the faster cleaving hammerhead is that it requires much lower levels of divalent metal ions (Mg2+) for activity. Both NMR and fluorescence techniques will be used to probe the effect of Mg2+ on the structure and dynamicsof these faster cleaving hammerhead ribozymes.
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