## 4 MazE-MazF: Autoregulation of a Chromosomally Encoded Type II Paradigm  
The MazF toxin encoded by _E. coli_ is the archetype of a broad family of sequence-specific endoribonucleases that cleave either free mRNA or ribosome-associated transcripts resulting in translation inhibition [23,24]. The activity of MazF is counteracted directly by the MazE antitoxin [51,52]. Conflicting evidence indicates that the MazE-MazF complex either is a primitive programmed cell death mechanism in bacteria, or that MazF toxicity instead induces reversible bacteriostasis from which cells can recover by subsequent production of MazE antitoxin as discussed elsewhere [3,9,53,54]. Antibiotics that inhibit transcription and/or translation prevent replenishment of the MazE antitoxin. As a consequence MazF, which is less proteolytically labile than MazE, is released to cause toxicity. Stressful conditions such as extreme amino acid starvation also inhibit _mazEF_ transcription. When uncharged tRNA species occupy the ribosomal A-site during amino acid starvation, the ReIA protein is activated and catalyzes synthesis of the alarmone guanosine tetraphosphate (ppGpp). Overproduction of ppGpp represses the _mazEF_ cassette by interfering with RNA polymerase function. The consequent decrease in MazE levels liberates the MazF toxin to act intracellularly [51]. Interestingly, the _mazG_ gene that is cotranscribed with _mazEF_ provides another level of control to MazF action. MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes dNTPs, thereby depleting ppGpp concentration. MazG activity is counteracted by the MazE-MazF complex. However, when amino acid starvation induces elevated ppGpp concentration with a concomitant reduction in expression of the _mazEFG_ module, MazG function is no longer blocked and ppGpp level decreases. Thus, the action of  
Figure 3: Organization of the promoter-operator regions upstream of the _ccdA-ccdB_, _mazE-mazF_, _relB-relE_, _ks-kid_, and _yefM-yoeB_ TA genes (top to bottom). \(-10\) and \(-35\) promoter motifs are coloured red and yellow, respectively. Repeat sequences are denoted by blue arrows; the sequences of these repeats differ among the different systems. The FIS binding site in the _mazE-mazF_ promoter region is shown as a hatched box. the MazE-MazF complex is balanced by the antagonistic activities of ppGpp that blocks _mazEFG_ transcription and by MazG which hydrolyzes the alarmone [55]. The _mazEFG_ cassette is preceded by two promoters whose relative strengths differ approximately ten-fold [51,56]. Transcription of the genes is autorepressed: MazE partially regulates both promoters but stronger repression is achieved by the MazE-MazF complex. Repression involves binding of the complex to an operator that comprises three related, adjacent repeat motifs that overlap the promoter region (Figure 3). By interacting with the first and second motifs or the second and third motifs, MazE-MazF may autorepress expression either to varying levels and/or in response to different physiological stimuli [56]. The MazE protein forms a homodimer within the MazE-MazF crystal structure, adopting a swapped-hairpin \(\beta\)-barrel structure with two extended C-terminal regions that interact with MazF. This structure identifies MazE as a member of the superfamily of DNA binding proteins typified by the transcription factor AprB. Operator recognition by MazE is thought to involve basic residues on the underside of the \(\beta\)-barrel element [52]. Interestingly, the factor for inversion stimulation (Fis) also binds to the _mazEFG_ promoter region (Figure 3) and modestly up-regulates _mazEFG_ expression [56]. Fis is a small, nucleoid-associated protein that binds to sites throughout bacterial chromosomes thereby regulating a wide array of genes, both directly by promoter interaction and indirectly by altering DNA structure during various growth stages [57,58]. Fis abundance varies dramatically during cell cycle progression [59].