The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in Enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by sitespecific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify cleavage targets for 11 VapCs from M. tuberculosis commencing with UV-crosslinking and deep sequencing. Remarkably, these VapCs are all site-specific endoribonucleases that cleave RNA targets that are essential for decoding at the ribosomal A-site. Ten VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translatio
