Structure Determination of a Large RNA by NMR Spectroscopy: The VS Ribozyme

University Of California-Davis, Davis CA

Investigators

Abstract

TECHNICAL: The project is aimed at the structural characterization of the catalytic element of VS RNA, an 881 nucleotide, single-stranded plasmid present in the mitochondria of certain natural strains of Neurospora. Most ribozymes recognize a complimentary single-stranded RNA through Watson-Crick base pairing; however, the VS ribozyme recognizes its substrate primarily through tertiary interactions. How this recognition occurs is not well understood, and it is clear that more structural information is needed to augment the wealth of biochemical data currently available. Due to the size of the VS ribozyme (~154 nucleotides) the project uses a modular approach. Initially the structures of isolated domains of the RNA, stem-loops as well as helix junctions, will be determined using standard NMR methodology. These structures and resonance assignments will then serve as starting points for the analysis of their conformation as part of the intact ribozyme using a combination of recently developed NMR techniques and specific/segmental labeling strategies. The results from these experiments will provide insight into how RNA tertiary structure influences the secondary structure of the building blocks and provide information about the domain structures in the functional ribozyme. In the last phase of the project residual dipolar couplings and information form other sources (NOEs, FRET studies, etc.) will be used to determine the global structure and cleavage mechanism of a functional ribozyme. NON-TECHNICAL: Ribozymes are RNA molecules with the capability to catalyze chemical reactions. This research is aimed at the structure determination of one such system: The Neurospora VS ribozyme. Multidimensional NMR spectroscopy will be used as the main tool to determine first the structures of isolated domains and then the global structure of the molecule. The information derived from the structures will provide insights into how RNA catalysis works on the molecular level.