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Thesis Advisor: Masayori Inouye, Ph.D.
Graduate Program in Biochemistry and Molecular Biology
Friday, October 24, 2008
mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. We have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to –mt7) and Staphylococcus aureus also contains one MazEF operon. The expression of the MazF-mt1,-mt3, -mt4, -mt6 and MazF-sa in E. coli will cause cell growth arrest. The in vivo and in vitro primer extension shows that MazF-mt1 preferentially cleaves era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA.
Using the phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate, we have also determined that MazF-mt3 cleaves RNA at UU CCU or CU CCU; MazF-mt7 at U¡CGCU and MazF-sa cleaves U ACAU ( indicates the cleavage site). Since pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis or S. aureus mRNAs that may be resistant or sensitive to MazF-mt3 and MazF-mt7 or MazF-sa cleavage. The pentad sequence of both MazF-mt3 and MazF-mt7 was found to be significantly underrepresented in the PE and PPE families in M. tuberculosis. The pentad sequence of MazF-sa is unusually highly abundant in the pathogenic adhesive factor, Srap.
We also found that the genes for MazF-mt1 and MazF-mt3 are co-localized with their cognate upstream genes in a putative operon. Their gene products are termed MazL-mt1 and MazL-mt3, respectively. Interestingly these proteins have very little homologies to E. coli MazE, the antitoxin for E. coli MazF. However, M. tuberculosis contains two independent operons encoding E. coli MazE homologues, MazE-mt1 and MazE-mt2, and each of them is co-localized with the gene for its cognate partner, MazJ-mt1 and MazJ-mt2, respectively. We have demonstrated that the expression of MazL-mt1 can suppress the toxicity of MazF-mt1. Consistent to this finding, the MazF-mt1 can bind to the MazL-mt1 in vitro to form a complex. We also demonstrated that the expression of MazL-mt1, -mt3 and MazE-mt1, -mt2 can suppress the toxicity of MazF-mt3. Their relations were also demonstrated by in vitro experiments. Furthermore, I found that MazE-mt1 or –mt2 and MazJ-mt1 or –mt2 also interact. Together, these results suggest that there is a sophisticated TA network in M. tuberculosis, which may be essential for tight regulation of the MazF toxicity in this pathogen, which may be important to stably maintain the dormancy of this pathogen in human tissues.