Archaeal box C/D sRNAs guide the 2?-O-methylation of target nucleotides in both ribosomal and tRNAs. These small non-coding RNAs are characterized by conserved terminal box C/D and internal C?/D? RNA motifs. Each RNA motif binds three core proteins to establish individual RNP complexes that catalyze the site-specific 2?-O-methylation of target nucleotides. Specificity of nucleotide modification is determined by target RNA base pairing with complementary sRNA D or D? guide sequences. The fifth target nucleotide upstream from the D or D? box within the guide:target RNA is then methylated by the core proteins. In vitro assembly of Methanocaldococcus jannaschii sR8 box C/D RNA with recombinant core proteins, L7, Nop56/58, and fibrillarin produces a methylation-competent sRNP complex. This model box C/D sRNP has now been used to determine the structural features of the guide:target RNA duplex that are important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was essential for nucleotide methylation. Mismatched bases within the guide:target RNA duplex also disrupted target nucleotide methylation. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA helix as target deoxy-oligonucleotides possessing a target ribonucleotide were not methylated. Methylation specificity at the base paired guide:target nucleotide was compromised by elevated Mg2+ concentrations. In high divalent cation concentrations, target nucleotides not hydrogen bonded to the guide nucleotide were nevertheless methylated. Interestingly, D and D? target RNAs were methylated to different levels when deoxynucleotides were inserted within the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These results suggested that structural features unique to the box C/D and C?/D? RNPs affect their nucleotide methylation capabilities. Finally, the ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested an intrinsic ability of this archaeal RNA:protein enzyme to unwind double-stranded target RNAs prior to nucleotide modification.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-08162006-142559 |
| Date | 23 August 2006 |
| Creators | Appel, Cathryn Denise |
| Contributors | A. Clay Clark, E. Stuart Maxwell, Linda Hanley-Bowdoin |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-08162006-142559/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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