RESUMEN
CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay.IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD, which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD, causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Sistemas de Lectura Abierta , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Biosíntesis de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Factor sigma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cyclic AMP (cAMP) and the cAMP receptor protein (cAMP-CRP) and CsrA are the principal regulators of the catabolite repression and carbon storage global regulatory systems, respectively. cAMP-CRP controls the transcription of genes for carbohydrate metabolism and other processes in response to carbon nutritional status, while CsrA binds to diverse mRNAs and regulates translation, RNA stability, and/or transcription elongation. CsrA also binds to the regulatory small RNAs (sRNAs) CsrB and CsrC, which antagonize its activity. The BarA-UvrY two-component signal transduction system (TCS) directly activates csrB and csrC (csrB/C) transcription, while CsrA does so indirectly. We show that cAMP-CRP inhibits csrB/C transcription without negatively regulating phosphorylated UvrY (P-UvrY) or CsrA levels. A crp deletion caused an elevation in CsrB/C levels in the stationary phase of growth and increased the expression of csrB-lacZ and csrC-lacZ transcriptional fusions, although modest stimulation of CsrB/C turnover by the crp deletion partially masked the former effects. DNase I footprinting and other studies demonstrated that cAMP-CRP bound specifically to three sites located upstream from the csrC promoter, two of which overlapped the P-UvrY binding site. These two proteins competed for binding at the overlapping sites. In vitro transcription-translation experiments confirmed direct repression of csrC-lacZ expression by cAMP-CRP. In contrast, cAMP-CRP effects on csrB transcription may be mediated indirectly, as it bound nonspecifically to csrB DNA. In the reciprocal direction, CsrA bound to crp mRNA with high affinity and specificity and yet exhibited only modest, conditional effects on expression. Our findings are incorporated into an emerging model for the response of Csr circuitry to carbon nutritional status. IMPORTANCE: Csr (Rsm) noncoding small RNAs (sRNAs) CsrB and CsrC of Escherichia coli use molecular mimicry to sequester the RNA binding protein CsrA (RsmA) away from lower-affinity mRNA targets, thus eliciting major shifts in the bacterial lifestyle. CsrB/C transcription and turnover are activated by carbon metabolism products (e.g., formate and acetate) and by a preferred carbon source (glucose), respectively. We show that cAMP-CRP, a mediator of classical catabolite repression, inhibits csrC transcription by binding to the upstream region of this gene and also inhibits csrB transcription, apparently indirectly. We propose that glucose availability activates pathways for both synthesis and turnover of CsrB/C, thus shaping the dynamics of global signaling in response to the nutritional environment by poising CsrB/C sRNA levels for rapid response.
Asunto(s)
Represión Catabólica , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genéticaRESUMEN
Toxin/antitoxin (TA) systems are the means by which bacterial cells become persistent; that is, those cells that are tolerant to multiple environmental stresses such as antibiotics by becoming metabolically dormant. These persister cells are responsible for recalcitrant infections. Once toxins are activated by the inactivation of antitoxins (e.g., stress-triggered Lon degradation of the antitoxin), many toxins reduce metabolism by inhibiting translation (e.g., cleaving mRNA, reducing ATP). The MqsR/MqsA TA system of Escherichia coli cleaves mRNA to help the cell withstand oxidative and bile acid stress. Here, we investigated the role of secondary structure and 5' mRNA processing on MqsR degradation of mRNA and found that MqsR cleaves only single-stranded RNA at 5'-GCU sites and that MqsR is equally active against RNA with 5'-triphosphate, 5'-monophosphate, and 5'-hydroxyl groups.