Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters.

Kolb, Annie; Busby, Stephen J. W.; Herbert, Michel; Kotlarz, Denise; Buc, Henri

doi:10.1002/j.1460-2075.1983.tb01408.x

Cited by 52 publications

(30 citation statements)

References 22 publications

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“…In the absence of cAMP, CRP failed to form the distinct complex, confirming that cAMP-CRP bound specifically to csrC DNA. The apparent dissociation constant (K d value) for cAMP-CRP binding to csrC DNA was 18 Ϯ 2 nM, similar to that of the lactose and galactose promoters (5 to 10 nM), which are known targets of cAMP-CRP binding (65). EMSA of CRP binding to csrA, uvrY, and csrD DNA in all cases resulted in the formation of diffuse complexes at high CRP concentrations (Ն100 nM), similarly to the results seen with csrB DNA, indicative of nonspecific binding (data not shown).…”

Section: Fig 2 Effects Of Camp-crp On Csrb and Csrc Levels And Decay mentioning

confidence: 68%

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

et al. 2016

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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 csrClacZ 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. IMPORTANCECsr (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.T he Csr (carbon storage regulator) or Rsm (repressor of stationary-phase metabolites) system is a widely conserved bacterial posttranscriptional regulatory system (1-3). Its components, their functions, and the mechanisms by which the central regulator of this system, CsrA or RsmA, affects gene expression have been studied primarily in Gammaproteobacteria (4-6). The...

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Section: Fig 2 Effects Of Camp-crp On Csrb and Csrc Levels And Decay mentioning

confidence: 68%

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

et al. 2016

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“…Interactions of RNA polymerase and CRP at mutant galpromoters We have previously isolated a series of gal promoter mutants which specifically block the activation of P1 because they stop the binding of cAMP-CRP (Busby et al, 1982Kolb et al, 1983). We have examined the interactions of polymerase with gal promoter DNA containing two such mutations: the p37 mutation, a GC to AT transition at -37 and the R503 deletion, which removes the gal sequence between -45 and -92.…”

Section: Resultsmentioning

confidence: 99%

On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions.

Spassky¹,

Busby²,

Buc³

1984

The EMBO Journal

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124

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“…In other catabolite-sensitive operons, the sequences in this region are required for efficient CAP binding. Removal of these sequences from the galactose operon blocks CAP action in vivo (10) and in vitro (34). The effect on araBAD was less dramatic, but three of the consensus base pairs were restored by upstream pBR322 sequences (Fig.…”

Section: Discussionmentioning

confidence: 98%

Repression and catabolite gene activation in the araBAD operon

Lichenstein¹,

Hamilton²,

Lee³

1987

J Bacteriol

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Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five-to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.The arabinose regulon is one of many gene systems in Escherichia coli that are modulated by catabolite gene activator protein (CAP) complexed with cyclic AMP (cAMP) (3,4,15,56,62). Mutations in adenyl cyclase (cya) or CAP (crp) genes give rise to Ara-phenotypes (72); it is not clear whether these Ara-phenotypes are due to inactivation of one or more catabolite-sensitive promoters of the arabinose regulon. The regulon consists of four operons, araBAD, araC, araE, and araFG; these are responsible for Larabinose catabolism, gene regulation, low-affinity transport, and high-affinity transport, respectively (7,21,23,35,37). In addition to positive control by CAP-cAMP, each operon is also regulated by AraC protein. Regulation of ara genes by AraC involves both positive and negative control mechanisms. AraC protein, plus the ligand L-arabinose, is required for activation of transcription at the promoters of the araBAD, araE, and araFG operons (35,42). In addition, AraC protein is also a repressor of araBAD (22) and araC (12,43,54) transcription, functioning to further reduce the level of araBAD in uninduced cells and autoregulate its intracellular level under both inducing and noninducing conditions.The CAP dependence of the arabinose regulon has been explored by using many techniques. Casadaban (12) demonstrated that a crp mutation reduced expression of araC. Kolodrubetz and Schleif (36) showed that a crp mutation reduced expression of the araE, araFG, and araBAD genes. The latter study was complicated by the fact that araE, araFG, and araBAD are positively regulated by AraC protein, which is itself sensitive to catabolite activation (12,29). Mutations in the high-affinity CAP-binding site between the araBAD and araC genes (Fig. 1) Philadelphia, PA 19101. base deletion in the CAP consensus sequence. The...

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Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters.

Cited by 52 publications

References 22 publications

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions.

Repression and catabolite gene activation in the araBAD operon

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