Targeted removal of the 16S rRNA anti-Shine–Dalgarno sequence by a Mycobacterium tuberculosis MazF toxin

The genome of the bacterial pathogen that causes tuberculosis, Mycobacterium tuberculosis (Mtb), encodes an inexplicably high number of type II toxin-antitoxin (TA) systems. Because this ancient pathogen has evolved to resist clearance by antibiotics and the host immune system, its TA systems are thought to participate in the survival of these stresses. Of the ∼70 Mtb type II TA systems, 10 MazEF family members have been previously identified, yet the precise cellular target of only one of these MazF toxins is known. Here, we demonstrate that the Rv3098A gene encodes an 11th MazF paralog in Mtb (MazF-mt11, MazF11). As with all MazF toxins, MazF-mt11 acts as a single-strand, sequence-specific endoribonuclease. We first performed primer extension on the large single-stranded MS2 enterobacteriophage RNA substrate after incubation with recombinant MazF-mt11 to identify a single toxin cleavage site between C↓A. We then further pinpointed the boundaries of the MazF-mt11 cleavage consensus sequence as C↓ACCU using Escherichia coli MORE (Mapping by Overexpression of an RNase in E. coli) RNA-seq. Finally, we enlisted 5'-OH RNA-seq to reveal 16S rRNA in the 30S ribosomal subunit as the only MazF-mt11 RNA target in mycobacteria. In fact, the single cleavage site in C↓ACCU maps just before the anti-Shine-Dalgarno sequence at the 3' end of 16S rRNA. Targeted removal of the anti-Shine-Dalgarno sequence by MazF-mt11 leads to nearly complete inhibition of protein synthesis, consistent with its important role in directing ribosomes to translation start codons in leadered mRNAs. The accompanying growth arrest phenotype suggests that MazF-mt11 may participate in establishment of the nonreplicating persistent state in Mtb.