# Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity
## 2 Families of Sequence-Specific Endoribonuclease TA Toxins
### MazF-ec  
MazF-ec is the toxin of the best studied Type II TA system, \(mazEF\) on the _E. coli_ chromosome [3; 20; 23]. MazF-ec recognizes the single-stranded RNA triplet sequence (ACA) and cleaves after the first A-residue (denoted as A'CA with \(\hat{}\) indicating the cleavage site) [3]. Pulse labeling experiments have shown that the production of a majority of the cellular proteins ceases when MazF-ec is expressed in vivo [52]. As almost all mRNA contain ACA triplets, the authors postulated that MazF-ec cleaves nearly all of the cellular mRNA in a ribosome-independent manner, thereby halting cellular protein synthesis. The continued production of a protein whose transcript is engineered to be devoid of ACA suggests that the protein synthetic process itself remains operational following MazF overproduction [52; 53]. While MazF-ec cleaves all available ACA sites in vitro, a recent RNA-seq analysis of the cellular RNA following MazF-ec overproduction identified a preferential digestion of ACA sites at or closely upstream of the AUG start codon to produce short-leaderless mRNA (ImRNAs) [32]. In addition to mRNA, the digestion of 16S rRNA by MazF was detected in recent studies [32; 54]. The ACA site at the 5\({}^{\prime}\) of helix 45 is prone to degradation by MazF-ec. Upon cleavage, 43 nucleotides from the 3\({}^{\prime}\) terminus, including the anti-Shing-Dalgarno (aSD) sequence, are removed from the 16S rRNA. The ribosome devoid of aSD is called the stress ribosome (70S\({}^{AA3}\)). Due to the lack of aSD, the stress ribosome cannot initiate translation of full-length mRNA [32]. However, the stress ribosomes remain assembled and retain their ability to translate mRNA that lacked SD (lmRNA). Originally, the stress ribosomes were postulated to translate specific sets of transcripts. However, later studies with _Mycobacteria_ identified an association of a variety of transcripts with the stress ribosomes [32; 54]. [PERSON] et al. have recently claimed that MazF and MqsR induce rRNA cleavages at multiple sites in vivo [55]. RNA-seq analysis identified that some cleavage had occurred at sequences that were not recognized by MazF and MqsR, suggesting an induction of one or more additional nucleases. These results also imply that MazF does not digest 16S rRNA within the fully-assembled 70S ribosome, but cleaves excess rRNA precursors. The extent of 16S rRNA digestion in vivo, as well as its physiological significance remain enigmatic. Earlier studies reported a lack of 16S rRNA digestion by MazF-ec, while later studies detected extensive digestion as discussed above [3; 54; 32]. [PERSON] et al. demonstrated the ability of MazF-expressing cells to synthesize a desired peptide from ACA-less transcripts [32; 52; 53]. Successful translations of ACA-less transcripts in MazF-producing cells seem impossible if cells are completely devoid of fully-assembled ribosomes with aSD [52; 53]. Fractions of complete 70S ribosomes present in MazF-expressing cells must be carefully examined in future studies. Structural studies of the MazF complex revealed that one MazE dimer binds two MazF dimers, forming a 2:4 hetero-hexameric complex [38]. The interaction between MazE and MazF is mediated bythe unstructured C-term extension of MazE, which binds to the dimer of MazF. The highly negative charge of the MazE C-term extension is thought to mimic RNA and binds the active site of MazF [3; 23]. Therefore, upon the binding of MazE, the catalytic activity of MazF is inhibited. There is a negative cooperativity between two RNA binding sites in the MazF homodimer [56]. The binding of one MazE to one of the two RNA binding sites within a MazF dimer efficiently inhibits the catalytic activity of both sites. Like other Type II TA systems, MazE by itself, as well as the MazEF complex function as transcriptional repressors of their own expression [57]. The 5' UTR of the _mazEF_ loci contains two promoters with alternating palindromes.