The stringent response is defined as the physiological changes elicited by amino acid starvation. Many of these changes depend on the regulatory nucleotide ppGpp (guanosine tetraphosphate) synthesized by RelA (ppGpp synthetase I), the relA-encoded protein. The second rel locus of Escherichia coli is called relBE and encodes RelE cytotoxin and RelB antitoxin. RelB counteracts the toxic effect of RelE. In addition, RelB is an autorepressor of relBE transcription. Here we reveal a ppGpp-independent mechanism that reduces the level of translation during amino acid starvation. Artificial overexpression of RelE severely inhibited translation. During amino acid starvation, the presence of relBE caused a significant reduction in the poststarvation level of translation. Concomitantly, relBE transcription was rapidly and strongly induced. Induction of transcription occurred independently of relA and spoT (encoding ppGpp synthetase II), but instead depended on Lon protease. Consistently, Lon was required for degradation of RelB. Replacement of the relBE promoter with a LacI-regulated promoter indicated that strong and ongoing transcription of relBE is required to maintain a proper RelB:RelE ratio during starvation. Thus relBE may be regarded as a previously uncharacterized type of stress-response element that reduces the global level of translation during nutritional stress.starvation ͉ activation of transcription ͉ relE ͉ relB
The Escherichia coli relBE operon encodes a toxin-antitoxin pair, RelE-RelB. RelB can reverse inhibition of protein synthesis by RelE in vivo. We have found that although RelE does not degrade free RNA, it cleaves mRNA in the ribosomal A site with high codon specificity. Among stop codons UAG is cleaved with fast, UAA intermediate and UGA slow rate, while UCG and CAG are cleaved most rapidly among sense codons. We suggest that inhibition of protein synthesis by RelE is reversed with the help of tmRNA, and that RelE plays a regulatory role in bacteria during adaptation to poor growth conditions.
Summary
RelE and ChpAK (MazF) toxins of Escherichia coli have previously been described as proteins that mediate efficient cell killing. We show here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate. Later induction of transcription of the antitoxin genes relB or chpAI fully reversed the static condition induced by RelE and ChpAK respectively. We also provide a mechanistic explanation for these findings. Thus, induction of relE transcription severely inhibited translation, whereas induction of chpAK transcription inhibited both translation and replication. Hence, most likely, lack of colony formation is due to inhibition of translation in the case of relE and inhibition of translation and/or replication in the case of chpAK. Consistent with this proposal, later induction of transcription of the cognate antitoxin genes simultaneously reversed cell stasis and the inhibitory effects of RelE and ChpAK on macromolecular syntheses. These results preclude that RelE and ChpAK mediate cell killing during the conditions used here. In vivo and in vitro analyses of a mutant RelE protein supported that inhibition of colony formation was due to inhibition of translation.
Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family. The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells. This review describes the genetics, molecular biology, and evolution of the hok gene family. The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail. Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts. The metastable structures prevent translation and antisense RNA binding during transcription. Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements. Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated. The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.
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