Open AccessInvolvement of cAMP‐CRP in transcription activation and repression of the pck gene encoding PEP carboxykinase, the key enzyme of gluconeogenesis
Author(s) -
Nakano Masahiro,
Ogasawara Hiroshi,
Shimada Tomohiro,
Yamamoto Kaneyoshi,
Ishihama Akira
Publication year - 2014
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12466
Subject(s) - promoter , biology , microbiology and biotechnology , transcription (linguistics) , e box , repressor , caat box , binding site , phosphoenolpyruvate carboxykinase , gene , transcription factor , camp receptor protein , gene expression , genetics , linguistics , philosophy
c AMP 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 c AMP ‐CRP associated at downstream CRP box‐2 plays as a repressor for pck transcription only in the presence of high levels of cAMP‐CRP.