The RNA World hypothesis describes a period of time during the origins of life in which RNA molecules performed all catalysis and were the only form of information storage. A great deal of evidence has been obtained in support of this hypothesis, however a few key demonstrations are lacking. The first demonstration is of a molecule capable of self-replication that could have plausibly arisen from the prebiotic soup. Previously in the Lehman Laboratory, a 198-nucleotide RNA was discovered that could be fragmented into as many as four pieces ranging from 39 - 63 nucleotides in length. When these pieces were incubated together in a test tube, they re-formed the necessary covalent bonds to regenerate the full-length 198-nucleotide RNA. Furthermore, the full-length RNAs were catalytically active and made copies of themselves from the remaining pieces in solution, providing a model system of self-replication. I was able to remove >10% of the total length of the RNA, which substantially reduced the catalytic activity of the full-length molecule. I discovered several mutations that restored catalytic activity by improved folding and increased catalytic rates using in vitro selection. A subset of these mutations was found to aid in the assembly of the shortened full-length RNA from smaller fragments than were possible in the original system, enhancing the prebiotic relevance of this system. A second demonstration to bolster the RNA World hypothesis would be showing that RNA is capable of harvesting energy from its environment by performing oxidation and reduction reactions. Again using in vitro selection, I have completed five rounds of selection geared towards identifying a ribozyme that reduces benzoic acid to benzaldehyde using Zn2+ and NADH. Results to date suggest the selection is working and it should be continued for another five to ten generations. Finally, I have discovered an RNA sequence that forms knots during transcription, a phenomenon heretofore undocumented in RNA. This new topology has implications for RNA stability by rendering RNA more resistant to hydrolysis, and could impact catalysis through formation of more complex, knotted active sites. Taken together, these findings have improved our understanding of RNA folding and catalysis, and the plausibility of the RNA World.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-1362 |
| Date | 01 January 2010 |
| Creators | Burton, Aaron Steven |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
Page generated in 0.0022 seconds