Systematic search for the Cra-binding promoters using genomic SELEX system.
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.
Shimada
Tomohiro
T
Nippon Institute for Biological Science, Division of Molecular Biology, Ome, Tokyo 198-0024, Japan.
Fujita
Nobuyuki
N
Maeda
Michihisa
M
Ishihama
Akira
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010751:Phosphopyruvate Hydratase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D056968:Lac Repressors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010751:Phosphopyruvate Hydratase; D012097:Repressor Proteins; C068837:FruR protein, Bacteria; D056968:Lac Repressors