Yonglong Zhang

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.

Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence‐specific and frame‐dependent mRNA cleavage

Toxin–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 5′ 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 5′AAA – G/A3′ sequences leading to rapid decay possibly facilitated by the mRNA degradosome.

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit

Bacterial toxin–antitoxin (TA) systems (or “addiction modules”) typically 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. TA systems have also been implicated in several clinically important phenomena: biofilm formation, bacterial persistence during antibiotic treatment, and bacterial pathogenesis. TA systems harbored by pathogens also serve as attractive antibiotic targets. To date, the mechanism of action of the majority of known TA toxins has not yet been elucidated. We determined the mode of action of the Doc toxin of the Phd-Doc TA system. Doc expression resulted in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication. However, Doc did not cleave mRNA as do other addiction-module toxins whose activities result in translation inhibition. Instead, Doc induction mimicked the effects of treatment with the aminoglycoside antibiotic hygromycin B (HygB): Both Doc and HygB interacted with 30S ribosomal subunits, stabilized polysomes, and resulted in a significant increase in mRNA half-life. HygB also competed with ribosome-bound Doc, whereas HygB-resistant mutants suppressed Doc toxicity, suggesting that the Doc-binding site includes that of HygB (i.e., helix 44 region of 16S rRNA containing the A, P, and E sites). Overall, our results illuminate an intracellular target and mechanism of TA toxin action drawn from aminoglycoside antibiotics: Doc toxicity is the result of inhibition of translation elongation, possibly at the translocation step, through its interaction with the 30S ribosomal subunit.

The Inhibitory Mechanism of Protein Synthesis by YoeB, an Escherichia coli Toxin

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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{\mathrm{f}}^{\mathrm{Met}}\) \end{document}. 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.

Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP

Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazF(Sa)) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazF(Sa) with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazF(Sa) specifically cleaves the RNA at a pentad sequence, U downward arrow ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazF(Sa) was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEF(Sa) TA module and the pathogenicity in S. aureus.

Inhibitory Mechanism of Escherichia coli RelE-RelB Toxin-Antitoxin Module Involves a Helix Displacement Near an mRNA Interferase Active Site

In Escherichia coli, RelE toxin participates in growth arrest and cell death by inducing mRNA degradation at the ribosomal A-site under stress conditions. The NMR structures of a mutant of E. coli RelE toxin, RelER81A/R83A, with reduced toxicity and its complex with an inhibitory peptide from RelB antitoxin, RelBC (Lys47-Leu79), have been determined. In the free RelER81A/R83A structure, helix α4 at the C terminus adopts a closed conformation contacting with the β-sheet core and adjacent loops. In the RelER81A/R83A-RelBC complex, helix α3* of RelBC displaces α4 of RelER81A/R83A from the binding site on the β-sheet core. This helix replacement results in neutralization of a conserved positively charged cluster of RelE by acidic residues from α3* of RelB. The released helix α4 becomes unfolded, adopting an open conformation with increased mobility. The displacement of α4 disrupts the geometry of critical residues, including Arg81 and Tyr87, in a putative active site of RelE toxin. Our structures indicate that RelB counteracts the toxic activity of RelE by displacing α4 helix from the catalytically competent position found in the free RelE structure.

Characterization of YafO, an Escherichia coli toxin.

YafO is a toxin encoded by the yafN-yafO antitoxin-toxin operon in the Escherichia coli genome. Our results show that YafO inhibits protein synthesis but not DNA or RNA synthesis. The in vivo [35S]methionine incorporation was inhibited within 5 min after YafO induction. In in vivo primer extension experiments with two different mRNAs, the specific cleavage bands appeared 11–13 bases downstream of the initiation codon, AUG, 2.5 min after the induction of YafO. An identical band was also detected in in vitro toeprinting experiments when YafO was added to the reaction mixture containing 70 S ribosomes and the same mRNAs even in the absence of tRNAfMet. Notably, this band was not detected in the presence of YafO alone, indicating that YafO by itself does not have endoribonuclease activity under the conditions used. The full-length mRNAs almost completely disappeared 30 min after YafO induction in in vivo primer extension experiments, consistent with Northern blotting analysis. Over 84% of [35S]methionine-tRNAfMet was released from the translation initiation complex at 5.43 μm YafO in vitro. We demonstrated that the 70 S ribosome peak significantly increased upon YafO induction, and when the 70 S ribosomes dissociated into 50 and 30 S subunits, YafO was found to be associated with 50 S subunits. These results demonstrate that YafO is a ribosome-dependent mRNA interferase inhibiting protein synthesis.

Staphylococcus aureus YoeB Homologues Inhibit Translation Initiation

YoeB is a bacterial toxin encoded by the yefM-yoeB toxin-antitoxin system found in various bacterial genomes. Here, we show that Staphylococcus aureus contains two YoeB homologues, both of which function as ribosome-dependent mRNA interferases to inhibit translation initiation in a manner identical to that of YoeB-ec from Escherichia coli.