Type of Document Dissertation Author Rashid, Rumana Author's Email Address firstname.lastname@example.org URN etd-11212005-141641 Title Structural and Biochemical Characterization of RNA-Guided Modification Enzymes Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Timothy M. Logan Committee Chair Lloyd M. Epstein Committee Member Michael S. Chapman Committee Member Robert H. Reeves Committee Member Keywords
- Box H/ACA
- Box C/D
Date of Defense 2005-11-17 Availability unrestricted AbstractAll functional RNAs contain post-transcriptionally modified nucleotides. 2’-O-ribose methylation and pseudouridylation are the two major types of modifications, which occur by box C/D and box H/ACA ribonucleoprotein (RNP) complexes, respectively. Generally, the guide RNA part binds to the complementary regions in the target RNAs but the actual catalysis is carried out by the protein part of the complex. In eukaryotes, these RNPs are called small nucleolar (sno)RNP complexes and in Archaea these are known as small (s)RNPs. The archaeal box C/D sRNP is a complex of three proteins: fibrillarin, Nop5p and L7Ae. Whereas, box H/ACA sRNP contains four proteins: Cbf5, Nop10, Gar1 and L7Ae.
During the last two decades significant progress has been achieved in identifying the components of these two classes of sRNPs but less is known about their mode of assembly, steps of assembly and catalysis processes. The work of this manuscript describes the basic mechanisms governing protein-protein, protein-RNA interactions within archaeal sRNP particles by employing biochemical and crystallographic approaches.
During the initial investigation, gel mobility shift assay was used to study A fulgidus box C/D RNP complex. It was established that L7Ae nucleates the whole complex by
changing the RNA conformation and subsequently binding with fibrillarin/Nop5p complex where Nop5p was observed to be the RNA binding protein. Next, we used gel shift assay, analytical ultracentrifugation, and in vitro methylation assay to study the assembly process of C/D RNP complex. Our results suggested that while a box C/D sRNP is capable of asymmetric assembly, the symmetries in both the box C/D RNA and in the fibrillarin/Nop5p complex are required for efficient catalysis.
Finally, a high-resolution crystal structure of Cbf5-Nop10-Gar1 protein complex was obtained from P furiosus. The structure captures a functional assembly state of the H/ACA sRNP particle and thus provides a mechanistic understanding on how this system works. Cbf5 shares major structural domains with other pseudouridine synthases, but displays structural differences consistent with its distinct function in RNA-guided pseudouridylation. In addition, we described the previously unknown structures of both Nop10 and Gar1, and their essential roles in pseudouridylation process.
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