The Varkud satellite (VS) ribozyme is a naturally occurring RNA molecule, found in the bread mold Neurospora, that catalyzes the self-cleavage and ligation reactions necessary for the life cycle of the VS RNA. It is the largest known member of the small self-cleaving ribozymes and the only one in this class for which the structure is not known at atomic resolution. Using single-molecule fluorescence resonance energy transfer (smFRET), we have studied how the molecule’s global dynamics influence its function as a catalyst1. However, to gain more detailed insights into the VS ribozyme’s three-dimensional structure, dynamics, and catalytic mechanism, a high-resolution X-ray crystal structure of the complete molecule is crucial. To this end, we are trying to crystallize the VS ribozyme using a novel method of purifying the ribozyme in its native state without subjecting it to denaturing conditions. By avoiding the harsh conditions of traditional purification by denaturing gel electrophoresis, which can cause the molecule to refold into erroneous structures, we hope to isolate a conformationally homogeneous population more conducive to crystallization and structural studies.
1. Pereira, M. J. B.; Nikolova, E. N.; Hiley, S. L.; Jaikaran, D.; Collins, R. A.; Walter, N. G., Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis. Journal of Molecular Biology 2008, 382, (2), 496-509.