Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent cleavage occurred at target sequences situated either in single- or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.     

Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.     

MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli

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.     

Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase

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 RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.     

Interference of mRNA function by sequence-specific endoribonuclease PemK

In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.     

Characterization of ChpBK, an mRNA interferase from Escherichia coli

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues, and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5'-or3'-side of the A residue in ACY sequences (Y is U, A, or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2',3'-cyclic phosphate group at the 3'-end of the 5'-end product and a 5'-OH group at the 5'-end of the 3'-end product in a manner identical to that of MazF.     

The discovery of mRNA interferases: implication in bacterial physiology and application to biotechnology

Escherichia coli contains a large number of suicide or toxin genes, whose expression leads to cell growth arrest and eventual cell death. This raises intriguing questions as to why E. coli contains so many toxin genes and what are their roles in bacterial physiology. Among these, MazF has been shown to be a sequence-specific endoribonuclease, which cleaves mRNAs at ACA sequences to completely inhibit protein synthesis. MazF is therefore called mRNA interferase. A number of other mRNA interferases with different cleavage specificities have been discovered not only in E. coli, but also in other bacteria including Mycobacterium tuberculosis. Induction of MazF in the cell leads to cellular dormancy termed quasi-dormancy. In spite of complete cell growth inhibition, cells in the quasi-dormant state are fully capable of energy metabolism, amino acids and nucleic acids biosynthesis and RNA and protein synthesis. The quasi-dormancy may be implicated in cell survival under stress conditions and may play a major role in pathogenicity of M. tuberculosis. The quasi-dormant cells provide an intriguing novel biotechnological system producing only a protein of interest in a high yield. MazF causing Bak-dependent programmed cell death in mammalian cells may be used as a tool for gene therapy against cancer and AIDS. The discovery of a novel way to interfere with mRNA function by mRNA interferases opens a wide variety of avenues in basic as well as applied and clinical sciences.     

Characterization of mRNA interferases from Mycobacterium tuberculosis

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. MazF-mt1 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.     

Characterization of the interactions within the mazEF addiction module of Escherichia coli

In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin. The mazEF system is known as an addiction module located on the Escherichia coli chromosome. MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex. MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression. Here we show that the binding of purified (His)6MazE to the mazEF promoter DNA was enhanced by MazF. The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF-(His)6 complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain. The ratio of MazE to MazF(His)6 in the MazE-MazF(His)6 complex is about 1:2. Because both MazE and MazF-(His)6 exist as dimers by themselves, the MazE-MazF-(His)6 complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)6 dimers. The interaction between MazE and MazF was also characterized with the yeast two-hybrid system. It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF. Site-directed mutagenesis at this region revealed that Leu55 and Leu58 play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA. The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF.     

MazG -- a regulator of programmed cell death in Escherichia coli

We have previously reported that mazEF, the first regulatable chromosomal 'addiction module' located on the Escherichia coli chromosome, downstream from the relA gene, plays a crucial role in the programmed cell death in bacteria under stressful conditions. It consists of a pair of genes encoding a stable toxin, MazF, and MazE, a labile antitoxin interacting with MazF to form a complex. The cellular target of MazF toxin was recently described to be cellular mRNA, which is degraded by this toxin. On the same operon, downstream to the mazEF genes, we found another open reading frame, which was called mazG. Recently, it was shown that the MazG protein has a nucleotide pyrophosphohydrolase activity. Here we show that mazG is being transcribed in the same polycistronic mRNA with mazEF. We also show that the enzymatic activity of MazG is inhibited by MazEF proteins. When the complex MazEF was added, the enzymatic activity of MazG was about 70% inhibited. We demonstrate that the enzymatic activity of MazG in vivo causes depletion of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation conditions. Based on our results, we propose a model in which this third gene, which is unique for chromosomal addiction systems, has a function of limiting the deleterious activity of MazF toxin. In addition, MazG solves a frequently encountered biological problem: how to avoid the persistence of a toxic product beyond the time when its toxicity is useful to the survival of the population.     

The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome

The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.     

Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway

In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.     

The Escherichia coli mazEF suicide module mediates thymineless death

In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its molecular mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P(2) is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.     

大肠埃希菌TA系统mazEF的研究进展

大部分细菌的遗传物质中含有毒素-抗毒素系统(TA)的遗传基因.mazEF是大肠埃希菌染色体上的一对毒素抗毒素基因,由毒素基因mazF和抗毒素基因mazE组成.其在细菌的生长调控和细胞程序性死亡中发挥了重要的作用.环境压力激活mazEF后,MazF可以通过对mRNA的剪切作用造成翻译停止.mazEF的存在可以增加细菌对环境压力的耐受性、保持细菌遗传物质的稳定、参与抗生素引起的细胞死亡、也在细菌的耐药性中发挥重要作用.     

Prokaryotic toxin-antitoxin stress response loci

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.     

Ribonucleases in bacterial toxin–antitoxin systems

Toxin–antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNA^fMet in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

ACA‐specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF‐ec) cleaves RNA at A and CA. To date, a large number of MazF homologs that cleave RNA at specific three‐ to seven‐base sequences have been identified from bacteria to archaea. MazF‐ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate‐binding site. Here, we investigated the role of the two loops in MazF‐ec, which are closely associated with the interface of the MazF‐ec dimer. We examined whether exchanging the loop regions of MazF‐ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF‐mx) and MazF from Mycobacterium tuberculosis (MazF‐mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF‐ec with loop 2 regions from either MazF‐mx or MazF‐mt3 created a new cleavage sequence at (A/U)(A/U)AA and C in addition to the original cleavage site, A and CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA and C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications. Proteins 2013. © 2012 Wiley Periodicals, Inc.