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.
Highlights
The central metabolism of carbon source catabolism for energy production consists of three pathways, the main glycolysis (Embden-Meyerhof-Parnas; EMP) pathway, pentose-phosphate (PP) pathway and tri-carboxylic-acid (TCA) cycle [1]
Since cAMP Receptor Protein (CRP) exhibits functional inter-conversion depending on the presence or absence of specific effector cAMP [11], we carried out Genomic SELEX in the presence and absence of cAMP
The gel pattern of DNA fragments isolated after Genomic SELEX in the absence of cAMP did not change from the original DNA mixture, indicating that little enrichment of specific DNA fragments
Summary
The central metabolism of carbon source catabolism for energy production consists of three pathways, the main glycolysis (Embden-Meyerhof-Parnas; EMP) pathway, pentose-phosphate (PP) pathway and tri-carboxylic-acid (TCA) cycle [1]. The CRP regulon includes the genes encoding the transporters and the catabolic enzymes of non-glucose sugars [9,11,12,13,15]. Expression of these genes is activated in the absence of glucose through functional conversion of CRP into an active form after interaction with an effector cAMP [17], which is synthesized by the membrane-bound adenylate cyclase. When cAMP-CRP binds DNA, it induces DNA bending of about 87O [16,22,23]
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