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Systematic search for the Cra‐binding promoters using genomic SELEX system
Author(s) -
Shimada Tomohiro,
Fujita Nobuyuki,
Maeda Michihisa,
Ishihama Akira
Publication year - 2005
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2005.00888.x
Subject(s) - promoter , biology , systematic evolution of ligands by exponential enrichment , gene , dna binding site , microbiology and biotechnology , genetics , binding site , transcription (linguistics) , transcription factor , genome , gene expression , rna , linguistics , philosophy
Cra (or FruR), a global transcription factor with both repression and activation activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra‐binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra‐binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hitherto‐unidentified genes. All six known promoters are repressed by Cra, but none of the activation‐type promoters were cloned after two cyles of SELEX, because the Cra‐binding affinity to the repression‐type promoters is higher than the activation‐type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra‐binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3‐phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the in vivo promoter strength assay using a newly developed TFP (two‐fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein.