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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.

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The consensus sequences of CRP binding.The binding sequences of cAMP-CRP were subjected to the Logo analysis for determinatin of the consensus sequences for the following samples: (A) the whole set of CRP targets (total 323 sequences) identified by Genomic SELEX screening in this study; (B) the set of CRP targets (165 sequences) that are listed in Regulon DB and identified by Genomic SELEX; (C) the set of CRP targets (129 sequences) that are listed in Regulon DB but not identified by Genomic SELEX; (D) the whole set of CRP targets (294 sequences) listed in Regulon DB.
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pone-0020081-g002: The consensus sequences of CRP binding.The binding sequences of cAMP-CRP were subjected to the Logo analysis for determinatin of the consensus sequences for the following samples: (A) the whole set of CRP targets (total 323 sequences) identified by Genomic SELEX screening in this study; (B) the set of CRP targets (165 sequences) that are listed in Regulon DB and identified by Genomic SELEX; (C) the set of CRP targets (129 sequences) that are listed in Regulon DB but not identified by Genomic SELEX; (D) the whole set of CRP targets (294 sequences) listed in Regulon DB.

Mentions: The consensus recognition sequences of CRP have been proposed after sequence analysis of the hitherto identified targets (for a review see [38]). As a test of the accuracy of Genomic SELEX screening for cAMP-CRP binding sequences, we searched for the CRP-box sequence using the whole set of 323 CRP-binding sequences from a total of 275 CRP targets (183 novel plus 92 known targets) identified in this study [note that some of the CRP targets carry multiple CRP-box sequences] (for details see Table S4). A collection of 500-bp sequences centered on each peak and using BioProspector (http://ai.stanford.edu/~xsliu/BioProspector/), which was successfully employed for identification of RutR box sequence [33], we identified the 16-bp sequence CRP-box motif for all 323 CRP-binding sites (Fig. 2A). The CRP box-consensus sequence, 5′-TGTGA-N6-TCACA-3′, agrees well with the hitherto identified [38], but indicates clearly that G at position 4 and C at position 13 of the CRP-box are highly conserved in good agreement of their key roles to exhibit the high affinity to CRP. In the Genomic SELEX screening herewith employed, a total of 92 known targets (47%) were identified from a total of 195 CRP targets deposited in Regulon RD. After separate analysis of CRP-box sequence for two groups of CRP targets from Regulon DB, it turned clear that the consensus CRP-box sequence is highly conserved for the CRP targets identified by Genomic SELEX (Fig. 2B). In contrast, the level of CRP-box sequence conservation is significantly lower for the group of unidentified CRP targets (Fig. 2C). This finding supports the usefulness of Genomic SELEX screening for quick identification of the whole set of regulation targets by cAMP-CRP with high accuracy.


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)

The consensus sequences of CRP binding.The binding sequences of cAMP-CRP were subjected to the Logo analysis for determinatin of the consensus sequences for the following samples: (A) the whole set of CRP targets (total 323 sequences) identified by Genomic SELEX screening in this study; (B) the set of CRP targets (165 sequences) that are listed in Regulon DB and identified by Genomic SELEX; (C) the set of CRP targets (129 sequences) that are listed in Regulon DB but not identified by Genomic SELEX; (D) the whole set of CRP targets (294 sequences) listed in Regulon DB.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105977&req=5

pone-0020081-g002: The consensus sequences of CRP binding.The binding sequences of cAMP-CRP were subjected to the Logo analysis for determinatin of the consensus sequences for the following samples: (A) the whole set of CRP targets (total 323 sequences) identified by Genomic SELEX screening in this study; (B) the set of CRP targets (165 sequences) that are listed in Regulon DB and identified by Genomic SELEX; (C) the set of CRP targets (129 sequences) that are listed in Regulon DB but not identified by Genomic SELEX; (D) the whole set of CRP targets (294 sequences) listed in Regulon DB.
Mentions: The consensus recognition sequences of CRP have been proposed after sequence analysis of the hitherto identified targets (for a review see [38]). As a test of the accuracy of Genomic SELEX screening for cAMP-CRP binding sequences, we searched for the CRP-box sequence using the whole set of 323 CRP-binding sequences from a total of 275 CRP targets (183 novel plus 92 known targets) identified in this study [note that some of the CRP targets carry multiple CRP-box sequences] (for details see Table S4). A collection of 500-bp sequences centered on each peak and using BioProspector (http://ai.stanford.edu/~xsliu/BioProspector/), which was successfully employed for identification of RutR box sequence [33], we identified the 16-bp sequence CRP-box motif for all 323 CRP-binding sites (Fig. 2A). The CRP box-consensus sequence, 5′-TGTGA-N6-TCACA-3′, agrees well with the hitherto identified [38], but indicates clearly that G at position 4 and C at position 13 of the CRP-box are highly conserved in good agreement of their key roles to exhibit the high affinity to CRP. In the Genomic SELEX screening herewith employed, a total of 92 known targets (47%) were identified from a total of 195 CRP targets deposited in Regulon RD. After separate analysis of CRP-box sequence for two groups of CRP targets from Regulon DB, it turned clear that the consensus CRP-box sequence is highly conserved for the CRP targets identified by Genomic SELEX (Fig. 2B). In contrast, the level of CRP-box sequence conservation is significantly lower for the group of unidentified CRP targets (Fig. 2C). This finding supports the usefulness of Genomic SELEX screening for quick identification of the whole set of regulation targets by cAMP-CRP with high accuracy.

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