Abstract
In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin–antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5′-end of the active mRNA. Here, we propose that this 5′-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin–antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances.
Highlights
In enterobacteria like Escherichia coli, DNA damage induces the SOS stress response genes, one of which encodes the toxin of the type I toxin–antitoxin (TA) locus tisB-istR1
We showed that r-protein S1 promotes 30S ribosomal subunit binding to the single-stranded standby site of the mRNA
Substitutions G to A at position (G54A), C to A at position (C55A), and G to C at position 57 (G57C) should prevent formation of the pseudoknot and translation of +42 tisB mRNA (Figure 1E). These three independent mutations were introduced into +42 tisB mRNA, and 30S initiation complex (30S-IC) formation tested by toeprint assays in comparison to wild-type +42 tisB mRNA as control
Summary
In enterobacteria like Escherichia coli, DNA damage induces the SOS stress response genes, one of which encodes the toxin of the type I toxin–antitoxin (TA) locus tisB-istR1. TisB is repressed by the master regulator LexA [1,2,3]. It encodes a pore-forming toxin that arrests growth through membrane depolarization and promotes the formation of persisters in response to stress [4,5,6,7]. The tisB primary transcript, denoted +1 tisB mRNA (Figure 1A), is translationally inactive since the tisB ribosome binding site (RBS) is structurally sequestered [10,11]. Despite an identically sequestered RBS, this mRNA is translationally active, and confers toxicity to cells when overexpressed [4,10]
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