Limits...
Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources.

Shimada T, Fujita N, Yamamoto K, Ishihama A - PLoS ONE (2011)

Bottom Line: Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites.Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration.One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

ABSTRACT
CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

Show MeSH

Related in: MedlinePlus

Identification of cAMP-CRP-binding sites on the E. coli genome.Genomic SELEX search of cAMP-CRP-binding sequences was performed using the standard procedure [28] in the presence (filled circles) and absence of cAMP (open circles). SELEX fragments were subjected to SELEX-chip analysis using the tilling DNA microarray as described previously (39). Regulation target genes of CRP were predicted from the location of CRP-binding sites (for details see text). The genes associated with the peaks at the cut-off level above 30 are indicated. When the CRP-binding site is located upstream of divergently transcribed genes, two genes are shown, being connected with a flash mark. Genes under light green background represent newly identified CRP targets while those under pale orange background represent the CRP targets listed in Regulon DB [27]. The level of CRP binding (Y-axis) represents the fluorescent intensity ratio between SELEX samples in the presence and absence of CRP.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105977&req=5

pone-0020081-g001: Identification of cAMP-CRP-binding sites on the E. coli genome.Genomic SELEX search of cAMP-CRP-binding sequences was performed using the standard procedure [28] in the presence (filled circles) and absence of cAMP (open circles). SELEX fragments were subjected to SELEX-chip analysis using the tilling DNA microarray as described previously (39). Regulation target genes of CRP were predicted from the location of CRP-binding sites (for details see text). The genes associated with the peaks at the cut-off level above 30 are indicated. When the CRP-binding site is located upstream of divergently transcribed genes, two genes are shown, being connected with a flash mark. Genes under light green background represent newly identified CRP targets while those under pale orange background represent the CRP targets listed in Regulon DB [27]. The level of CRP binding (Y-axis) represents the fluorescent intensity ratio between SELEX samples in the presence and absence of CRP.

Mentions: For identification of the whole set of binding sequences by CRP, we then subjected the mixture of SELEX fragments to the newly developed DNA tilling array analysis (SELEX-chip) [29], [30]. Genomic SELEX fragments obtained with or without the effector cAMP were labeled with Cy5 while the original DNA library was labeled with Cy3. Two sets of the mixture of fluorescent-labeled samples were hybridized separately to the DNA tilling microarray (Oxford Gene Technology, Oxford, UK) [39]. For elimination of the bias of library DNA, the ratio of fluorescence intensity bound to each probe between the test sample and the original library DNA was measured, and plotted against the corresponding position along the E. coli genome (Fig. 1). On the DNA tilling array used, the 60 b-long probes are aligned along the E. coli genome at 105 bp-intervals, and therefore approximately 300 bp-long SELEX fragments should bind to two or more consecutive probes. This criterion was employed for identification of positive peaks of CRP binding (or elimination of false-positive peaks).


Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources.

Shimada T, Fujita N, Yamamoto K, Ishihama A - PLoS ONE (2011)

Identification of cAMP-CRP-binding sites on the E. coli genome.Genomic SELEX search of cAMP-CRP-binding sequences was performed using the standard procedure [28] in the presence (filled circles) and absence of cAMP (open circles). SELEX fragments were subjected to SELEX-chip analysis using the tilling DNA microarray as described previously (39). Regulation target genes of CRP were predicted from the location of CRP-binding sites (for details see text). The genes associated with the peaks at the cut-off level above 30 are indicated. When the CRP-binding site is located upstream of divergently transcribed genes, two genes are shown, being connected with a flash mark. Genes under light green background represent newly identified CRP targets while those under pale orange background represent the CRP targets listed in Regulon DB [27]. The level of CRP binding (Y-axis) represents the fluorescent intensity ratio between SELEX samples in the presence and absence of CRP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105977&req=5

pone-0020081-g001: Identification of cAMP-CRP-binding sites on the E. coli genome.Genomic SELEX search of cAMP-CRP-binding sequences was performed using the standard procedure [28] in the presence (filled circles) and absence of cAMP (open circles). SELEX fragments were subjected to SELEX-chip analysis using the tilling DNA microarray as described previously (39). Regulation target genes of CRP were predicted from the location of CRP-binding sites (for details see text). The genes associated with the peaks at the cut-off level above 30 are indicated. When the CRP-binding site is located upstream of divergently transcribed genes, two genes are shown, being connected with a flash mark. Genes under light green background represent newly identified CRP targets while those under pale orange background represent the CRP targets listed in Regulon DB [27]. The level of CRP binding (Y-axis) represents the fluorescent intensity ratio between SELEX samples in the presence and absence of CRP.
Mentions: For identification of the whole set of binding sequences by CRP, we then subjected the mixture of SELEX fragments to the newly developed DNA tilling array analysis (SELEX-chip) [29], [30]. Genomic SELEX fragments obtained with or without the effector cAMP were labeled with Cy5 while the original DNA library was labeled with Cy3. Two sets of the mixture of fluorescent-labeled samples were hybridized separately to the DNA tilling microarray (Oxford Gene Technology, Oxford, UK) [39]. For elimination of the bias of library DNA, the ratio of fluorescence intensity bound to each probe between the test sample and the original library DNA was measured, and plotted against the corresponding position along the E. coli genome (Fig. 1). On the DNA tilling array used, the 60 b-long probes are aligned along the E. coli genome at 105 bp-intervals, and therefore approximately 300 bp-long SELEX fragments should bind to two or more consecutive probes. This criterion was employed for identification of positive peaks of CRP binding (or elimination of false-positive peaks).

Bottom Line: Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites.Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration.One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

ABSTRACT
CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

Show MeSH
Related in: MedlinePlus