RelE/ParE-like RNase toxin RelS cleaves  anti-Shine-Dalgarno (anti-SD) core region
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 TA system in M. tuberculosis. We show that the RelS toxin is highly toxic in M. tuberculosis and is efficiently 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. Biochemical investigation of the toxic mechanism revealed that RelS is an atypical RelE/ParE-like RNase toxin that efficiently inhibits translation by targeting the 30S ribosomal subunit, specifically cleaving the 16S rRNA between positions C1520 and U1521, a unique site within the anti-Shine-  Dalgarno (anti-SD) core region. This work further highlights the antiSD region as a hot spot for deleterious RNase toxins in this major pathogen and demonstrates that there remains a greater arsenal of TA systems still to explore within mycobacteria.