Master of Science
Physiology and Biophysics
Janina P. Lewis, Ph.D.
Carlos Escalante, Ph.D.
Renfeng Li, Ph.D.
Carmen Sato-Bigbee, Ph.D.
BIOCHEMICAL ANALYSIS OF PUTATIVE RNA-BINDING PROTEINS IN PORPHYROMONAS GINGIVALIS
By Jacob Mirpanah
Bachelor of Arts in Biology, University of Virginia, 2015
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology and Biophysics at Virginia Commonwealth University
Virginia Commonwealth University, 2018
Major Director: Janina P. Lewis, Ph.D., Philips Institute for Oral Health Research
RNA-Binding Proteins (RBPs) play important regulatory roles in countless cellular processes. Often via the induction of a structural change in RNA’s secondary structure, RBPs are known to modulate protein expression through post-transcriptional regulation. In Escherichia coli, such RBPs have been thoroughly studied and shown to display differential expression throughout the bacterial life cycle; suggesting their importance in prompting different events in the bacterial cell. Further, RNA- and DNA-recognition domains have been characterized in proteins Hub as well as RBP in E.coli and their nucleic acid ligands sequenced. It was our aim to extend this level of information to Hub and RBP as they exist in a main etiological agent in periodontal disease; Porphyromonas gingivalis. Using quantitative PCR expression analysis, we saw a general upregulation of both proteins in the logarithmic phase as compared to the stationary phase. This upregulation was most pronounced in PG0627, the putative analog of E. coli RBP in P. gingivalis. Further, Electrophoretic Mobility Shift Assays suggest sequence specific interaction of PG0627 to RNA. Uninhibited and Inhibited mobility assays seem to confirm that PG0627 binds with great specificity to a conserved RNA sequence. Quantitative measure of interaction came in the form of fluorescence anisotropy, which produced a Dissociation Constant (Kd) of approximately 53 nM; suggesting a high degree of affinity. Lastly, a mutant was generated in order to produce high quality RNA libraries to be sequenced through the Illumina MiSeq system. Sequencing data is still incoming.
© Jacob Mirpanah
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Available for download on Sunday, May 07, 2023