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MAZF TOXINS FROM MYCOBACTERIUM TUBERCULOSIS TARGET THE TRANSLATION MACHINERY

by
Jason M. Schifano
B.S., Montclair State University - 2007

Thesis Advisors: Nancy A. Woychik, Ph.D.
Graduate Program in Microbiology & Molecular Genetics

RWJMS Research/School of Public Health Building
Conference Room 258, Piscataway

Tuesday, August 5, 2014
11:00 a.m.


Abstract

Mycobacterium tuberculosis contains a remarkably large number of toxin-antitoxin (TA) systems, gene pairs that are suggested to participate in establishing latent tuberculosis or adapting to stress conditions in the host. Eleven TA systems belong to the mazEF family, encoding the intracellular toxin MazF and its inhibitor, antitoxin MazE. Although the physiological roles of mazEF loci in M. tuberculosis are poorly understood, there are striking parallels between the “quasi-dormant” state of Escherichia coli cells upon MazF expression and the slow growth and dormancy of M. tuberculosis during latent infection. MazF toxins are endoribonucleases that arrest protein synthesis and growth by cleaving single-stranded RNA at specific and unique 3- to 7-nt sequences. Because MazF orthologs did not appear to cleave rRNA or tRNA in early studies, these toxins were labeled “mRNA interferases.” However, the view that MazF toxins exclusively target mRNA has been challenged by a recent study demonstrating that E. coli MazF cleaves 16S rRNA. Our work has further disputed this “mRNA interferase” model by showing that three M. tuberculosis MazF orthologs cleave rRNA or tRNA at specific sites. We first determined that MazF-mt3, MazF-mt6, and MazF-mt9 each recognize a unique 5-nt RNA sequence. We also found that MazF-mt6 cleaves 23S rRNA in the ribosomal A site, which is sufficient to inhibit translation. By applying a novel variation of RNA-seq that we developed, we found that MazF-mt3 cleaves 23S rRNA at the same location as MazF-mt6, but also cleaves within the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Removal of the aSD sequence raises the possibility that MazF-mt3, like E. coli MazF, reprograms ribosomes to preferentially translate leaderless mRNAs. Finally, our RNA-seq approach revealed that MazF-mt9 cleaves several tRNAs that contain its cleavage motif. Overall, our findings suggest that rRNA, tRNA, and mRNA are all targets of MazF toxins and support a model in which MazF toxins alter translation in distinct ways – either through its potent shutdown, by cleaving specific subpopulations of mRNA, or by reprogramming ribosomes to selectively translate leaderless mRNAs. This differential impact on translation might provide insight into the maintenance of so many seemingly redundant genes in M. tuberculosis.


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