# Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity
## 2 Families of Sequence-Specific Endoribonuclease TA Toxins
### MazF Homologues from M. tuberculosis  
M. tuberculosis contains nine copies of MazF, each with a distinctive sequence specificity (Table 1). The primer extension of _era_ mRNA in vivo determined that MazF-mt1 cleaves between the U and A-residue of CU'ACC [21]. The result was confirmed by in vitro digestion using purified MazF-mt1. Another minor cleavage sequence (UU'ACA) was also identified by in vitro assay. The in vitro digestion of shorter synthetic RNA revealed a flexible substrate range of MazF-mt1. The study with 15-base RNA demonstrated that the consensus sequence is U'AC, while a study using an 11-base RNA showed that any single base in UAC can be changed to another base and still be digested [21]. Similar flexibility was also observed for MazF-mt7. Most cleavage occurs with U'CGCU with one or two base alterations [45]. Although all cleavable sequences include a third G-residue, less stringent specificity has been observed for the other positions.  
Recent RNA-seq-based screening of the entire cellular mRNA revealed that the cleavage sequence for MazF-mt3 is also five bases long, and the recognition sequence is U'CCUU [26]. MazF-mt3 is also found to digest 23S and 16S rRNA [26]. Digestion of 23S rRNA occurs at the U'UCCU sequence within the single-stranded area of the helix/loop 70. This region of 23S rRNA serves multiple important functional roles of the ribosome. The region is crucial for ribosome assembly, as a single mutation in this sequence abolishes the association between 50S and 30S ribosomal subunits [59]. The region is also essential for the interaction with tRNA and with the ribosome recycling factor RRF [60]. As the recognition sequence is localized in the region that is crucial for ribosome assembly, the digestion by MazF-mt3 must lower the number of fully-functional ribosomes in the cell. Additionally, MazF-mt3 also digests 16S rRNA. Cleavage of 16S rRNA occurs at the anti-Shine-Dalgarno sequence [26]. Such loss is postulated to cause the selective translation of leaderless mRNA in _M. tuberculosis_ in a similar manner as 16S rRNA digestion by MazF-ec in _E. coli_. Although significant digestion has been detected with both in vivo and in vitro experiments, the digestion efficiency in vivo seems to be weak, as a majority of rRNA remains intact after 1 h of MazF-mt3 expression [26].  
While the original biochemical study of MazF-mt6 using _era_ mRNA appeared to show that this enzyme has a non-stringent sequence specificity, a recent RNA-seq-based study revealed that the consensus sequence is UU'CCU [21; 24]. MazF-mt6 also has activity towards 23S rRNA [24]. It digests the recognition sequence in helix/loop 70 of 23S rRNA [24]. This UUCCU signature is conserved in ribosomes from many taxa, suggesting its functional importance. In vitro protein synthesis of mRNA lacking a UUCCU motif is still blocked by the addition of MazF-mt6, indicating that MazF-mt6 inhibits translation not only via digestion of mRNA, but also due to rRNA digestion [24]. The ectopic expression of MazF-mt6 reduces the amount of full-length 23S rRNA, demonstrating that the digestion of rRNA also occurs in vivo. The authors further showed that MazF-mt6 cleaves 23S rRNA in the 50S ribosome, but does not digest 23S rRNA in the 70S ribosome [24]. Their results suggest that MazF-mt6 inhibits the assembly of 70S ribosomes, while it has no effect on pre-existing 70S complexes.  
With the RNA-seq analysis, MazF-mt9 is shown to digest two specific types of tRNA, as well as mRNA at UU'U [25]. The majority of the tRNA in _M. tuberculosis_, totaling 45, are resistant to cleavage by MazF-mt9, despite the presence of a recognition sequence. Digestion is limited to the UUU within the single stranded regions of tRNA. Only two types of tRNA are digested by MazF-mt9; the D-loop of \(\mathrm{tRNA^{Pro14}}\) and the single-stranded region within the \(\mathrm{tRNA^{Lys43}}\) anticodon [25].