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43875-AC4
Nils G. Walter , University of Michigan
Normal 0 false false false MicrosoftInternetExplorer4 During the past few years it has been recognized that non-coding RNAs play a much wider role in many aspects of the replication, processing, modification and regulation of genetic information. Non-coding RNAs of complex tertiary structure whose structure-function relationships have been well studied include the self-cleaving ribozymes, in which catalytic activity depends directly on structure and can be measured without the need for reporter molecules or other macromolecular interactions. The VS ribozyme is the catalytic motif embedded in VS RNA, which is found in mitochondria of geographically and genetically divergent species of the bread mold Neurospora . Similar to a retrotransposon, VS RNA is converted into a DNA plasmid through enzymatic action of a reverse transcriptase encoded on the coexisting V retroplasmid. The reverse transcriptase requires a circular monomeric VS RNA template to generate the VS plasmid, which in turn is transcribed by mitochondrial RNA polymerase into multimeric VS RNA copies. Self-cleavage and re-ligation by the embedded VS ribozyme to regenerate circular monomeric VS RNA is thus an important feature of satellite replication in the fungal host. The large VS ribozyme is therefore a biologically important, non-protein catalyst that we set out to study by single molecule fluorescence resonance energy transfer (smFRET) and x-ray crystallography as described in the PRF proposal. Richard Collins from the University of Toronto , the discoverer of the VS ribozyme, has been our collaborator on these projects. The VS ribozyme provides a model system to address the relationship between the structural dynamics and function of non-coding RNA. 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 readily assayed in vitro. Using smFRET we found that the wild type ribozyme (WT) populates three distinct global conformations as characterized by the distance between the cleavage site in stem-loop I and the active site in helix V. These conformations consist of a high FRET state (H) with a distance of 35 Ǻ, a mid FRET state (M) of 55 Ǻ, and a low state (L) of 96 Ǻ. The rates of interconversion between these three conformations were determined by applying Hidden Markov modeling to the collected traces. The predominant transitions are between the H and M states. Bulk solution assays showed that our WT ribozyme is also fully catalytically active, implying that the observed structural dynamics is linked to the activity of the ribozyme.
We hypothesized that the H state encompasses formation of a catalytically essential kissing loop interaction between stem loops I and V. To test this hypothesis we introduced a single mutation that abolishes this interaction as well as catalytic activity. Consistent with our hypothesis, the H state is no longer observed and instead replaced by much longer lived M and L states. Furthermore, we found that a compensatory mutation almost completely rescues the H state as well as cleavage activity.
Taken together, our data led us to propose a hierarchical folding pathway towards catalysis. A correctly folded II-III-VI junction is a prerequisite for the VS ribozyme to access the catalytically critical H state. Kinetic modeling based on the measured cleavage rate constants in bulk solution and the transition rate constants derived at the single molecule level revealed that an additional kinetic barrier beyond formation of the H state must be traversed before catalysis can take place, i.e., the kissing loop interaction is only partially rate-limiting. We further propose a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. A manuscript on our findings was recently published in J. Mol. Biol. , see citation .
The second major aim of this PRF grant is the crystallization of the VS ribozyme. A critical step in crystallization is the large scale purification of a homogeneous sample of correctly folded molecules. We have made big strides in this direction by developing a novel native purification strategy. We are currently further optimizing this protocol to yield still higher purity material for crystallization trials. We are planning to publish this technique by year’s end.
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