Ribosomal RNA cleavage by the previously unidentified RelS-RelI toxin-antitoxin system controls growth of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems use diverse strategies to control bacterial growth and represent attractive therapeutic targets to fight pathogens. Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, encodes one of the largest repertoires of TA systems. Here, we applied a bioinformatic pipeline to predict candidate TA systems in mycobacterial genomes and identified Rv2663-Rv2664 (RelS-RelI) as a previously undetected system in M. tuberculosis. We show that the RelS toxin is highly toxic and is inhibited by a unique antitoxin, RelI. The 1.70 Å X-ray crystallographic structure of RelS:RelI shows an unprecedented heterooctameric quaternary TA complex formed by paired tetramers. In each tetramer, RelS toxins are held at each end of a RelI antitoxin dimer. RelI binds across the putative catalytic center of RelS, resulting in occlusion of essential putative target-binding residues. Investigation of the toxic mechanism revealed that RelS is an atypical RelE/ParE-like RNase toxin that inhibits translation by targeting the 30S ribosomal subunit, specifically cleaving the 16S ribosomal RNA between positions C1520 and U1521, a unique site within the anti-Shine-Dalgarno (anti-SD) core region. This work further highlights the anti-SD region as a hot spot for RNase toxins and extends the arsenal of TA systems harnessed by this major pathogen.