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Box C/D RNAs are non-coding small RNAs that guide methylation and processing of ribosomal RNAs. All snoRNAs are associated with specific proteins in snoRNP particles. The four core proteins identified in yeast are Nop1p (nucleolar protein 1 or Fibrillarin), Nop58p (also known as Nop5p), Nop56p, and Snu13p (small nuclear protein 13). In the archaeal organism Archaeoglobus fulgidus (AF), these four proteins correspond to AF Fibrillarin, AF Nop5p(homologue of Nop56p/Nop58p), and AF L7Ae. Previous biochemical and crystallographic studies have identified the network and the order of interactions among the four components however, no insight has been obtained on structural changes that take place during the step-wise assembly of the particles. Steady-state fluorescence resonance energy transfer (FRET) was used to monitor the conformational changes of a model box C/D RNA during sRNP assembly and substrate binding. Significant RNA conformational changes were observed at each assembly step that was dependent on structural integrities of both the proteins and the box C/D RNA. Steady-state FRET experiments show direct evidence that the kink-turn structure formed by the box C/D RNA is induced by the binding of L7Ae. The conformational change due to the binding of a Nop5p/Fibrillarin complex requires dimerization of the complex, as binding of the double mutant Nop5p/Fibrillarin complex at a 100:1 protein:RNA molar ratio failed to display the same level of decrease in the FRET efficiency as observed for the wild-type protein. Steady-state FRET results on the assembly of sRNPs using the box C/D (A/C mutant) RNA show a minimal disruption on the RNA conformational change upon Nop5p/Fibrillarin binding, despite the fact that L7Ae binds to only one box C/D motif. These protein-induced RNA conformational changes explain the obligated order of assembly of the box C/D sRNP particles and support an "induced-fit" model for box C/D sRNP assembly. These findings also raised a testable hypothesis that the box C/D RNA conformation stabilized by bound proteins facilitates specific recognition of the target RNA.
A Thesis submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Master of Science.
Includes bibliographical references.
Hong Li, Professor Directing Thesis; Nancy L. Greenbaum, Committee Member; Sanford A. Safron, Committee Member.
Florida State University
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