Type of Document	Dissertation
Author	Moore, Terrie Luong
URN	etd-05062005-184404
Title	Biophysical and Biochemical Investigation of an Archaeal Box C/D SRNP: RNA-Protein Interactions of a Kink Turn RNA within the Functional Enzyme
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Hong Li Committee Chair John Dorsey Committee Member Lloyd Epstein Committee Member Timothy Logan Committee Member
Keywords	L7Ae Kink Turn RNA
Date of Defense	2005-05-03
Availability	unrestricted
Abstract Box C/D snoRNPs catylze the specific 2’O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most are associated with proteins and protein binding may nucleate the assembly of other proteins onto the RNA. Dissecting the structure and biochemical properties of box C/D snoRNPs may not only help in understanding the function of the modifications, but may also give insight into the role of kink turn RNAs in RNP assembly. An archaeal box C/D RNA embedded within the intron of pre-tRNATrp from Archaeglobus Fulgidus(AF) that guides two modifications in the tRNA was used as the model for the investigation of three complexes: L7Ae-box C/D RNA, L7Ae-box C’/D’ RNA, and the entire box C/D sRNP. Extensive crystallization trials resulted in crystals for each complex. A crystal structure of the box C/D RNA-L7Ae complex was determined to 2.7Ĺ and shows the box C/D sequences do form a kink turn. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct kink turn RNAs revealed a conserved RNA-protein interface suggesting a conformational “adaptability” of the kink turn RNAs in binding L7Ae homologs. NMR characterization of L7Ae-box C/D RNA and L7Ae-box C’/D’ RNA interactions suggests a structural change in the RNAs upon binding L7Ae and the RNAs may be dynamic structures that do not form stable kink turns alone. The underlying differences in primary and secondary structures of the kink turns may lead to different tertiary structures and dynamic behavior in kink turn RNAs that may confer specificity of L7Ae homologs for different kink turn RNAs. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.
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