# Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity
## 1 Introduction
In all tested cases, sequence-specific ribonucleases can only cleave single-stranded RNA and do not cleave RNA/RNA or RNA/DNA  
Figure 1: Type II and III toxin-antitoxin (TA) systems. Toxins and antitoxin genes are represented by blue and orange thick arrows. Wavy lines represent mRNA that are transcribed from each module. In Type II TA systems, toxins and antitoxins are co-transcribed. Antitoxins form complexes with toxin proteins under preferred growth conditions. Antitoxins are degraded by stress-inducible proteases, such as Lon. Antitoxin and the TA complex can negatively regulate expression by binding to their own promoter region. In Type III TA systems, antitoxin RNA is processed by toxins that have nuclease activity. Both processed and unprocessed antitoxins remain bound to the toxin’s active site, inhibiting toxins from digesting other cellular RNAs. duplexes [3; 21]. The accessibility of the recognition sequence to the enzyme also seems crucial, as the sequences that are located within a secondary structure or buried within a large protein-RNA complex (e.g., ribosome) are not susceptible to cleavage [24; 25; 26]. The length of the cleavage sequence of MazF homologues varies from three to seven bases [1; 3; 27; 28]. For example, MazF-ec recognizes a triplet, ACA [3]. MazF homologues from Gram-positive _Staphylococcus aureus_ and _Bacillus subtilis_ cleave UACAU [27; 29; 30; 31]. MazF-hw from archaea _Haloquadratum walsbyi_ recognizes a seven-base sequence (UUACUCA) [28]. Relatively long RNA (e.g., MS2 RNA) has often been used as a substrate to analyze the sequence specificity of ribonucleases that recognize bases longer than five. However, because of the limited number of pentamers represented in one type of RNA substrate, identifying the exact consensus sequence could not be achieved in some cases [21; 24]. Recently, the RNA-seq-based method of detecting cleavage sites in all cellular RNAs (MORE RNA-seq) has been adopted. Due to the extensive sequencing depth, the technique allows for the refinement of the recognition sequences, as well as for the detection of novel RNA targets, such as tRNA, rRNA and tmRNA [24; 26]. The position and the frame of cleavage sequences within the mRNA seem to provide another layer of specificity for digestion in vivo. [PERSON] and colleagues demonstrated that MazF-ec cleaves ACA at or near the AUG start codon, producing leaderless mRNA (lmRNA) [32]. Furthermore, MazF-ec digests ACA sites only in the reading frame, while ACA sites that occurred out of the frame were not cleaved [33]. Coincidently, ribosome-dependent ribonucleases YoeB and RelE also showed a similar reading-frame-dependent cleavage preference [13; 17; 34]. _E. coli_ RelE preferentially digests a stop codon (UAG) within the ribosomal A site [13]. _E. coli_ and _S. aureus_ YoeB cleaves mRNA at three bases downstream of the initiation codon without sequence specificity [17; 34]. The digestion of MazF with reading-frame dependency suggests that although these enzymes can cleave RNA in a ribosome independent manner, the majority of in vivo digestion would occur in association with the ribosome. Preferential digestion near translational initiation sites also suggests the differential importance of translation initiation, elongation and termination processes on ribonuclease digestion. [PERSON] and colleagues postulated that the binding of MazF-ec to an extracellular-death-factor (EDF)-like element in the ribosomal protein bS1 is important for the RNA cleavage activity of MazF [33]. Further research is needed to decipher whether all of the sequence-specific ribonucleases cleave predominantly at the 5\({}^{\prime}\) and/or 3\({}^{\prime}\) end of mRNA and if it is indeed associated with a ribosome in vivo. While earlier in vivo digestion studies of sequence-specific ribonucleases were mainly focused on the digestion of mRNA, recent studies have shown that some toxins can also digest other types of RNA, such as rRNA, tRNA, sRNA and tmRNA [24; 25; 26; 27; 32; 35].