# Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity
## 1 Introduction  
Toxin-antitoxin (TA) systems are ubiquitous modules found in almost all sequenced bacterial genomes [1; 2]. Toxins are small polypeptides that negatively regulate cellular growth by inhibiting essential cellular processes, such as cell division, DNA replication, protein translation and membrane integrity [3; 4; 5; 6; 7]. Many genomes carry multiple copies of TA pairs, often each differing in the mechanisms of growth inhibition and expression patterns [1; 2; 8]. The transcription of a toxin gene is often coupled with its cognate antitoxin with these two genes typically forming an operon. Toxin activity is blocked by antitoxins when approximately an equivalent amount of toxin and antitoxin is present in a cell. Lowering the antitoxin concentration allows for the increase in free toxins, which leads to the arrest of cell growth. TA systems are classified into six subgroups based on the type of antitoxin (e.g., protein or RNA) and the mechanism of how antitoxin neutralizes toxin activity [9]. For example, in Type II TA systems, antitoxin proteins sequester toxins via physical interactions (Figure 1). Preferential digestion of antitoxin proteins by stress-inducible proteases frees toxins to exert their toxicity. In Type III TA systems, sRNA antitoxin is both a substrate and the inhibitor of the cognate endoribonuclease toxin. In type V TA systems, the antitoxin is a nuclease that specifically digests toxin mRNA to repress expression [10]. The vast majority of known TA toxins compromise protein translation [1; 11]. TA toxins inhibit translation through multiple mechanisms, including sequence-specific digestion of RNAs (rRNA, mRNA, tRNA, sRNA and/or mRNA), ribosome-dependent cleavage of mRNA, non-specific digestion of mRNA and the inhibition of ribosome assembly [1; 12; 13; 14]. Ribosome-dependent endoribonucleases, such as RelE, YoeB and YafO, do not cleave mRNA in the absence of ribosomes, as toxins themselves have zero or little nuclease activity [15; 16; 17; 13]. Upon association with a ribosome, toxins induce mRNA cleavage within the ribosomal A site immediately downstream of the start codon, or at 11 to 13 bases downstream of the initiation codons [4; 13; 17; 18]. Toxin YafQ of DinJ-YafQ is capable of mRNA cleavage in the absence of ribosome; however, sequence- and frame-specific cleavage occurs only in the presence of ribosomes [19]. In contrast, ribosome independent nucleases digest RNA in a sequence-specific manner even in the absence of ribosomes. While studies of ribosome-dependent inhibitions of translation and non-specific digestion of mRNA also have scientific merit, this review will focus specifically on the discovery and studies of toxins with ribosome-independent, sequence-specific ribonuclease activity. MazF from _Escherichia coli_ (MazF-ec) is the first TA toxin to have been identified as a sequence-specific ribonuclease [3; 20]. The original methods employed to characterize MazF-ec have been adapted to characterize a myriad of TA toxins with similar activities [3; 21; 22]. The initial discovery of MazF-ec inhibiting protein synthesis was achieved by monitoring the incorporation of \({}^{35}\)S-labeled methionine into newly-synthesized peptides [3; 22]. Upon ectopic expression of MazF-ec, the synthesis of proteins halts immediately [3]. In contrast, the rates of DNA replication and RNA synthesis do not change for a prolonged period, indicating that the direct target of MazF-ec is protein synthesis. Furthermore, Northern blot analysis confirmed the immediate disappearance of cellular mRNA following MazF-ec expression [3]. MALDI-mass spectrometry analysis of the product of MazF-ec-digested mRNA confirmed that MazF-ec is an endoribonuclease that specifically cleaves at the phosphodiester linkage [23]. The specific cleavage sites of MazF-ec and other endoribonucleases have been characterized by the in vivo expression of toxins followed by the primer extension of extracted RNA and/or by in vitro digestion of synthesized RNA by purified toxin protein [3; 20].