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

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 hithertounidentified 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 repressiontype 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.

Novel regulation targets of the metal-response BasS–BasR two-component system of Escherichia coli

“…Genomic SELEX screening of BasR-binding sequences was carried out using the improved procedure (Shimada et al, 2005). A mixture of DNA fragments of the E. coli K-12 W3110 genome was prepared after sonication of purified genome DNA, and cloned into a multi-copy plasmid, pBR322.…”

“…To begin the process of identifying the BasR regulon, we tried to identify the whole set of target promoters under the direct control of BasR using the improved Genomic SELEX screening system (Shimada et al, 2005), in which purified His-tagged BasR was mixed with a collection of E. coli genome fragments of 200-300 bp in length in the presence of acetyl phosphate, which phosphorylates BasR in the absence of BasS kinase. Phosphorylated BasR-bound DNA fragments were affinity-purified for the identification of BasR recognition sequences.…”

“…The predicted regulation targets of BasR include three groups of genes: group 1 includes a set of genes for the synthesis and modulation of membrane structure, such as arnB (LPS modification) and rfaB (LPS galactosyltransferase); group A Genomic SELEX search of phosphorylated BasR-binding sequences was performed using the standard procedure (Shimada et al, 2005). DNA fragments isolated by SELEX screening were subjected to mapping on the genome using a tiling DNA microarray (Shimada et al, 2011a, b;Teramoto et al, 2010).…”

“…The SELEX-chip method was developed for quick mapping of transcription factor-bound DNA segments across the E. coli genome using a DNA tiling array (Teramoto et al, 2010). The Genomic SELEX screening system has been successfully employed for identification of the whole set of regulation targets against a number of E. coli transcription factors, such as RstA (Ogasawara et al, 2007a), PdhR (Ogasawara et al, 2007b), RutR (renamed from YdcC) (Shimada et al, 2007), NemR (renamed from YdhM) (Umezawa et al, 2008), Dan (renamed from YgiP) (Teramoto et al, 2010), LeuO (Shimada et al, 2009, 2011a, Cra (Shimada et al, 2005(Shimada et al, , 2011c and CRP (Shimada et al, 2011b). BasR binding in vitro to the predicted BasSR targets was examined in detail by gel shift and DNase I footprinting assays, while regulation in vivo was examined by Northern blot analysis.…”

“…To identify the binding sequences within the entire E. coli genome for phosphorylated BasR, we employed both SELEX (systematic evolution of ligands by exponential enrichment)-clos and SELEX-chip systems of the improved 'Genomic SELEX' screening system in vitro (Shimada et Ishihama, 2010). In the SELEX-clos system, transcription factor-bound DNA fragments are cloned and sequenced (Shimada et al, 2005). The SELEX-chip method was developed for quick mapping of transcription factor-bound DNA segments across the E. coli genome using a DNA tiling array (Teramoto et al, 2010).…”