Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-11032008-154759 |
| Date | 13 November 2008 |
| Creators | Wakely, Heather |
| Contributors | Bonham-Smith, Peta C. |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-11032008-154759/ |
| Rights | restricted, 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 University of Saskatchewan 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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