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1 Nippon Institute for Biological Science, Division of Molecular Biology, Ome, Tokyo 198-0024, Japan
2 Meiji University, Faculty of Agriculture, Kawasaki, Kanagawa 214-8571, Japan
3 National Institute of Technology and Evaluation, Genome Analysis Center, Nishihara, Shibuya, Tokyo 151-0066, Japan
4 Hosei University, Faculty of Engineering and Research Center for Micro-Nano Technology, Koganei 184-8584, Tokyo 184-8584, Japan
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.
* Correspondence: E-mail: aishiham{at}nibs.or.jp
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