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Jung Ho Park
M.S., New Jersey Institute of Technology – 2005
Thesis Advisors: Masayori Inouye, Ph. D.
Graduate Program Cell & Developmental Biology
CABM, Room 010
Wednesday, April 18, 2012
mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. A MazF homologue from Bacillus subtilis (MazF-bs) has been shown to inhibit cell growth when induced in Escherichia coli. In the first research section of my dissertation, I determined that MazF-bs is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of the secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites.
In the second section, in order to further investigate the RNA binding mechanism of mRNA interferases, two methods, computational structure modeling and random or site directed mutagenesis, were used for MazF-ec (MazF from E. coli) and MazF-bs. First, from the structure model of RNA-MazF-ec complex, I proposed that RNA-sequence recognition specificities for MazF-ec were controlled by the flexible loop regions and showed that the exchanging and replacing of the loop regions of MazF-ec with those from other MazF homologues or poly-glycine affects the RNA cleavage specificity. Interestingly, the exchanging of loop 2 with loop 2 regions from either MazF-mx (MazF in Myxococcus xanthus) or MazF-mt3 (MazF in Mycobacterium tuberculosis) resulted in a new cleavage sequence at (A/U)(A/U)A^C in addition to the original cleavage site, ACA. Additionally, from other methods, it was also proposed that S19, R25, and H59 may be the catalytic and RNA recognition residues in MazF-bs protein.
In the third research section, MazF-ec was explored to develop a novel approach for preventive and therapeutic treatment of infection by single-stranded RNA viruses. The protease activity of the virus was used to activate a toxic protein to prevent viral infection instead of targeting these proteases to inhibit viral infection. I engineered the MazE-MazF fusion protein in which a C-terminal fragment of antitoxin MazE was fused to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease) or Factor Xa. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF. The intramolecular regulation of MazF toxicity by proteases may thus provide a novel approach for preventive and therapeutic treatment of infection by single-stranded RNA viruse