Single molecule characterization of the Varkud Satellite ribozyme and bulk native purification of non-coding RNA

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in all organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be assayed in vitro. We utilize single molecule fluorescence resonance energy transfer (FRET) microscopy to show that the VS ribozyme exhibits previously unobserved dynamic and hierarchical folding into an active structure. Readily reversible kissing loop formation combined with the slow observed cleavage of the VS RNA lead to the discovery of an additional downstream barrier that must be overcome before the VS RNA can cleave its immediate upstream substrate.;An alternative substrate available to the VS ribozyme is the distal downstream substrate that is part of the next monomer of the multimeric replication intermediate of VS RNA. Recent evidence suggests this is the preferred substrate in vivo. The role that structural dynamics play in this preference was unknown and is investigated here. Single molecule FRET studies of an RNA that models this alternate substrate selection show that in contrast to the previously observed global dynamics, these VS RNA molecules exhibit comparably little global dynamics. These differences in folding characteristics are correlated to the large difference in cleavage rates. These observed differences in structural dynamics, are consistent with a proposed pathway in vivo that minimizes exposure of the ends of linear VS RNA to the exonucleases of the host cell.;A prerequisite to many experiments is an RNA preparation procedure whose end-product is the target RNA in the same structure as found in the cell. To this end, a novel native RNA purification procedure has been developed. This protocol yields a pure target RNA whose structure more closely resembles what is found in nature than what the current standard RNA purification methods achieve.