A complete understanding of protein synthesis requires description of the cyclical motions and interactions that occur in ribosomal RNA and transfer RNA during translation. The purpose of the following research was to gain understanding of the nature of these motions and interactions, and to develop new tools to measure how ligands alter the conformational flexibility of RNA during the steps of initiation, elongation and termination. Different tRNA substrates were bound to the E. coli 70S to simulate the arrangement of the tRNA-ribosomal complex before and after peptide bond formation, and different UV-induced 16S rRNA-tRNA photocrosslinks were produced in these complexes, illustrating that the 16S rRNA P-site undergoes local deformations during elongation. A statistical study was undertaken to understand the nature of conformational states in the 30S ribosomal subunit. Using the lists of observed UVB/C- and UVA-s4U-induced crosslinks and the T. thermophilus 30S X-ray crystal structure, frequencies were compared to a number of geometrical parameters demonstrating that crosslink formation requires substantial RNA motions. In addition, the results show that the restricted pattern of crosslink formation in E. coli 16S rRNA is due to the overall rigidity of the 30S subunit outside of the active site. One consequence of these conclusions is that potocrosslinking rates depend on the ease of inter-nucleotide conformational movements. This was exploited in a study that used the temperature response of the rate constant for the UVA-induced photo-crosslink between s4U8 X C13 in E. coli tRNA to determine tRNA geometry and internal energy. The rate constants followed Arrhenius behavior in their dependence on temperature, and this allowed calculation of the activation energy associated with the conformational rearrangement necessary to bring the photoreactive bonds together. The experiments show that changes in the tRNA on ribosomes can be uncovered by photocrosslinking, that RNA mobility occurs by transient conformational changes, and describe a new technique than can quantitatively measure the internal energy associated with these conformational movements.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-06042006-151722 |
| Date | 19 June 2006 |
| Creators | Huggins, Bruce Wayne |
| Contributors | Dennis Brown, Stuart Maxwell, Paul Wollenzien |
| 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-06042006-151722/ |
| 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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