Porphyromonas gingivalis (P. gingivalis) is a gram-negative, anaerobic bacterium recognized as a major player in progression of periodontal disease. P. gingivalis survives in the oral cavity while being exposed to dynamic environmental conditions such as pH, temperature, nutrient availability and host immune responses such as oxygen tension and nitrosative stress. Survival and pathogenesis of P. gingivalis in the oral cavity require mechanisms to regulate gene expression in response to the extracellular signals. Little is known about the regulatory mechanisms of P. gingivalis in the oral cavity, so it is important to investigate and characterize these regulatory mechanisms. Adaptation to environmental cues using riboregulation is a significant mechanism for post-transcriptional regulation in bacteria. Using bioinformatics, we have identified a putative RNA-binding protein in P. gingivalis: RBP. Bioinformatic studies have led to the selection of HUβ and HUα nucleoid associated proteins as controls for RNA binding. I hypothesize that the candidate proteins RBP, HUβ and HUα bind RNA in P. gingivalis. The first aim is to show that RBP, HUβ and HUα bind RNA. Using electrophoretic mobility shift assays with IRE RNA and synthesized RNA motifs, I have confirmed that the proteins do bind RNA. The second aim is to isolate and sequence the P. gingivalis RNA that bind to RBP, HUβ and HUα. I have isolated the RNAs that bound the proteins and determined identity of the RNA using high throughput sequencing. Finally, I have identified an antibody that specifically binds RBP to use for in vivo immunoprecipitation of RNA-protein complexes from P. gingivalis. In conclusion RBP, HUβ and HUα are novel RNA binding proteins in P. gingivalis, and further investigation of these proteins is necessary to understand the mechanisms of gene regulation in P. gingivalis.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4526 |
| Date | 01 January 2014 |
| Creators | Dwyer, Holly |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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