Involvement of cAMP-CRP in transcription activation and repression of the<i>pck</i>gene encoding PEP carboxykinase, the key enzyme of gluconeogenesis

Nakano, Masahiro; Ogasawara, Hiroshi; Shimada, Tomohiro; Ishihama, Akira

doi:10.1111/1574-6968.12466

Cited by 16 publications

(16 citation statements)

References 32 publications

(51 reference statements)

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“…Repression by cAMP-CRP can be accomplished in a number of ways. For example, CRP can bind at a location close to the promoter and directly interfere with transcription initiation or elongation (57,72). Alternatively, CRP can prevent binding of an activator (52) or can activate transcription from a promoter and indirectly lead to repression of transcription from an overlapping divergent promoter (73,74).…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

et al. 2016

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“…AFM was performed in air with the tapping mode of a Multimode AFM (Veeco, CA, USA) and an Olympus silicon cantilever (OMCL-AC160TS-W2; Olympus, Tokyo, Japan). Imaging was carried out according to the method described previously (55) with a slight modification: the glutaraldehyde cross-linking step was omitted. Images were analyzed using the NANOSCOPE software.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of bacterial gene rearrangement: SprA-catalyzed precise DNA recombination and its directionality control by SprB ensure the gene rearrangement and stable expression of spsM during sporulation in Bacillus subtilis

Abe

Takamatsu

Sato

2017

Nucleic Acids Research

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A sporulation-specific gene, spsM, is disrupted by an active prophage, SPβ, in the genome of Bacillus subtilis. SPβ excision is required for two critical steps: the onset of the phage lytic cycle and the reconstitution of the spsM-coding frame during sporulation. Our in vitro study demonstrated that SprA, a serine-type integrase, catalyzed integration and excision reactions between attP of SPβ and attB within spsM, while SprB, a recombination directionality factor, was necessary only for the excision between attL and attR in the SPβ lysogenic chromosome. DNA recombination occurred at the center of the short inverted repeat motif in the unique conserved 16 bp sequence among the att sites (5΄-ACAGATAA/AGCTGTAT-3΄; slash, breakpoint; underlines, inverted repeat), where SprA produced the 3΄-overhanging AA and TT dinucleotides for rejoining the DNA ends through base-pairing. Electrophoretic mobility shift assay showed that SprB promoted synapsis of SprA subunits bound to the two target sites during excision but impaired it during integration. In vivo data demonstrated that sprB expression that lasts until the late stage of sporulation is crucial for stable expression of reconstituted spsM without reintegration of the SPβ prophage. These results present a deeper understanding of the mechanism of the prophage-mediated bacterial gene regulatory system.

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“…AFM observation of DNA–protein complexes was performed essentially as described [82] using two forms of the rrnE operon segment, probe A1 and probe A2 (for the probe list see Table 1), which were prepared by PCR using a set of primers (S2 Table). DNA–protein mixtures were incubated in the binding buffer for 30 min at 37°C, and after cross-linking of DNA–protein by treatment with 0.05% glutaraldehyde for 30 min at 4°C, the samples were diluted with the dilution buffer and directly spotted onto a freshly cleaved mica substrate pretreated with 10 mM spermidine.…”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli

et al. 2016

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Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3’ proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.

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Involvement of cAMP-CRP in transcription activation and repression of thepckgene encoding PEP carboxykinase, the key enzyme of gluconeogenesis

Cited by 16 publications

References 32 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

Mechanism of bacterial gene rearrangement: SprA-catalyzed precise DNA recombination and its directionality control by SprB ensure the gene rearrangement and stable expression of spsM during sporulation in Bacillus subtilis

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli

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