Systematic search for the Cra-binding promoters using genomic SELEX system

Genes Cells. 2005 Sep;10(9):907-18. doi: 10.1111/j.1365-2443.2005.00888.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Consensus Sequence
  • Dose-Response Relationship, Drug
  • Electrophoretic Mobility Shift Assay
  • Escherichia coli Proteins / genetics*
  • Gene Expression Regulation, Bacterial
  • Genome, Bacterial
  • Genomic Library*
  • Genomics / methods*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Molecular Sequence Data
  • Open Reading Frames
  • Phosphopyruvate Hydratase / genetics
  • Promoter Regions, Genetic / genetics*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Transcription, Genetic

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Repressor Proteins
  • FruR protein, Bacteria
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Phosphopyruvate Hydratase