title: Involvement of cAMP-CRP in transcription activation and repression of the pck gene encoding PEP carboxykinase, the key enzyme of gluconeogenesis.
abstract: cAMP receptor protein (CRP) is the best characterized global regulator of Escherichia coli. After genomic SELEX screening, a total of minimum 378 promoters have been identified as its regulation targets on the E. coli genome. Among a number of promoters carrying two CRP-binding sites, several promoters carry two CRP-binding sites, one upstream but another downstream of transcription initiation sites. The regulatory role of downstream CRP site remains unsolved. Using the pck gene encoding phosphoenolpyruvate carboxykinase as a model promoter, we analyzed the role of CRP-associated downstream of the transcription initiation site. Gel shift assay and AFM observation indicate that CRP binds to both the promoter-distal site (CRP box-1) at -90.5 and the site (CRP box-2) at +13.5 downstream of transcription initiation site. The binding affinity is higher for CRP box-1. Roles of two CRP sites were examined using in vitro transcription assay and in vivo reporter assay. In both cases, transcription repression was observed in the presence of high concentrations of CRP. Taken together, we propose that cAMP-CRP associated at downstream CRP box-2 plays as a repressor for pck transcription only in the presence of high levels of cAMP-CRP.
authors.affiliation: Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ogasawara
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Shimada
authors.forename: Kaneyoshi
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Akira
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D063185:Epigenetic Repression / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005820:Genetic Testing; D005943:Gluconeogenesis / Q000235:genetics*; D018625:Microscopy, Atomic Force; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
chemical_list: D002373:Cyclic AMP Receptor Protein; D019764:Phosphoenolpyruvate Carboxykinase (ATP)
keywords: Escherichia coli; cAMP receptor protein CRP; pck promoter; transcription regulation