Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems. The cellular targets of these toxins are quite diverse and include DNA replication, mRNA stability, protein synthesis, cell-wall biosynthesis, and ATP synthesis. These toxins are co-expressed and neutralized with their cognate antitoxins from a TA (toxin-antitoxin) operon in normally growing cells. Antitoxins are more labile than toxins and are readily degraded under stress conditions, allowing the toxins to exert their toxic effect. Presence of at least 33 TA systems in Escherichia coli and more than 60 TA systems in Mycobacterium tuberculosis suggests that the TA systems are involved not only in normal bacterial physiology but also in pathogenicity of bacteria. The elucidation of their cellular function and regulation is thus crucial for our understanding of bacterial physiology under various stress conditions.

The mqsR gene has been shown to be positively regulated by the quorum-sensing autoinducer AI-2, which in turn activates a two-component system, the qseB-qseC operon. This operon plays an important role in biofilm formation in Escherichia coli. However, its cellular function has remained unknown. Here, we found that 1 base downstream of mqsR there is a gene, ygiT, that is co-transcribed with mqsR. Induction of mqsR caused cell growth arrest, whereas ygiT co-induction recovered cell growth. We demonstrate that MqsR (98 amino acid residues), which has no homology to the well characterized mRNA interferase MazF, is a potent inhibitor of protein synthesis that functions by degrading cellular mRNAs. In vivo and in vitro primer extension experiments showed that MqsR is an mRNA interferase specifically cleaving mRNAs at GCU. The mRNA interferase activity of purified MqsR was inhibited by purified YgiT (131 residues). MqsR forms a stable 2:1 complex with YgiT, and the complex likely functions as a repressor for the mqsR-ygiT operon by specifically binding to two different palindromic sequences present in the 5-untranslated region of this operon.

SUMMARY MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. How MazF specifically recognizes its mRNA target and carries out cleavage and how the formation of the MazE-MazF complex inactivates MazF remain unclear. We present crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis. The structure of MazF in complex with uncleavable UUdUACAUAA RNA substrate defines the molecular basis underlying the sequence-specific recognition of UACAU and the role of residues involved in the cleavage through site-specific mutational studies. The structure of the heterohexameric (MazF)2-(MazE)2-(MazF)2 complex in Bacillus subtilis, supplemented by mutational data, demonstrates that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites.

RNA interference mediated by RNA such as antisense RNA, short interfering RNA and micro RNA is well documented to regulate specifi c gene expression at the level of messenger RNA . However, RNA interference mediated by proteins has not been reported. Here we identify the MazF-hw mRNA interferase from a superhalophilic archaeon that cleaves RNA at a specifi c seven-base sequence (UUACUCA). This sequence was found unusually abundant in the mRNAs for rhodopsin transcription activator and some membrane proteins of the archaeon, suggesting that the expression of these proteins is regulated by MazF-hw. When all of the seven-base cleavage sites in essential genes in Escherichia coli were eliminated, the cells were no longer sensitive to MazF-hw, demonstrating that specifi c gene expression can be regulated by a sequence-specifi c mRNA interferase. These fi ndings demonstrate that mRNA interference can be mediated not only by RNA but also by proteins to effectively silence specifi c gene expression in cells.