Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.

MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5-end of ACA sequences, yielding a 2,3-cyclic phosphate at one side and a free 5-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNArestriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.

YoeB is a toxin encoded by the yefM-yoeB antitoxin-toxin operon in the Escherichia coli genome. Here we show that YoeB, a highly potent protein synthesis inhibitor, specifically blocks translation initiation. In in vivo primer extension experiments using two different mRNAs, a major band was detected after YoeB induction at three bases downstream of the initiation codon at 2.5 min. An identical band was also detected in in vitro toeprinting experiments after the addition of YoeB to the reaction mixtures containing 70 S ribosomes and the same mRNAs, even in the absence of tRNA f Met . Notably, this band was not detected in the presence of YoeB alone, indicating that YoeB by itself does not have endoribonuclease activity under the conditions used. The 70 S ribosomes increased upon YoeB induction, and YoeB was found to be specifically associated with 50 S subunits. Using tetracycline and hygromycin B, we demonstrated that YoeB binds to the 50 S ribosomal subunit in 70 S ribosomes and interacts with the A site leading to mRNA cleavage at this site. As a result, the 3-end portion of the mRNA was released from ribosomes, and translation initiation was effectively inhibited. These results demonstrate that YoeB primarily inhibits translation initiation.

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazFmt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.Most bacteria contain "suicidal" or "toxic" genes whose expression leads to growth arrest and eventual death upon exposure to stress. These toxin genes are usually coexpressed with their cognate antitoxin genes present in the same operon (1, 2). The Escherichia coli chromosome contains six such operons called toxin-antitoxin (TA) 2 systems. Of these, the MazE (antitoxin)/MazF (toxin) system is one of the most extensively characterized TA systems, and MazF has been shown to be a sequence-specific endoribonuclease that cleaves at ACA sequences in mRNAs (3, 4). Thus, MazF is an mRNA interferase, and its induction causes the effective inhibition of protein synthesis leading to cell growth arrest. However, MazF-induced cells fully retain cellular metabolic activities, including ATP production and amino acid and nucleotide synthesis, as well as RNA and protein synthesis. Therefore, MazF-induced cells are capable of synthesizing a protein, if the gene encoding this protein is devoid of ACA sequences (4). This metabolically active dormant state caused by mRNA interferase induction is called "quasidormancy" (4) and has important implications in the physiology of various pathogenic bacteria, including persistent multidrug resistance.Notably, Mycobacterium tuberculosis, one of the most devastating human pathogens, contains nearly forty TA systems on its genome, among which nine have been shown to be homologous to E. coli MazF (5). It has been proposed that the TA modules play important roles in bacterial survival and formation of stable persisters in adverse conditions. These phenomena are essential for the persistence of M. tuberculosis. Persisters are the few rare pre-existing bacterial cells in a culture at any growth phase that are not growing and are intrinsically resistant to antibiotics by virtue of their subdued metabolism (6, 7).In the present paper, we have characterized two of the MazF homologues from this pathogen, wh...

SummaryToxin-antitoxin (TA) systems on the chromosomes of free-living bacteria appear to facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. They have been implicated in several clinically important phenomena -bacterial persistence during antibiotic treatment, biofilm formation and bacterial pathogenesis -and serve as attractive new antibiotic targets for pathogens. We determined the mode of action of the YafQ toxin of the DinJ-YafQ TA system. YafQ expression resulted in inhibition of translation, but not transcription or replication. Purified YafQ exhibited robust ribonuclease activity in vitro that was specifically blocked by the addition of DinJ. However, YafQ associated with ribosomes in vivo and facilitated rapid mRNA degradation near the 5Ј end via cleavage at AAA lysine codons followed by a G or A. YafQ(H87Q) mutants lost toxicity and cleavage activity but retained ribosome association. Finally, LexA bound to the dinJ-yafQ palindrome and triggered module transcription after DNA damage. YafQ function is distinct from other TA toxins: it associates with the ribosome through the 50S subunit and mediates sequence-specific and frame-dependent mRNA cleavage at 5Ј AAA -G/A 3Ј sequences leading to rapid decay possibly facilitated by the mRNA degradosome.