Curation Information

Publication: Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by FNR, NarL and NarP.;Partridge JD, Browning DF, Xu M, Newnham LJ, Scott C, Roberts RE, Poole RK, Green J;Microbiology (Reading, England) 2008 Feb; 154(Pt 2):608-18 [18227264]
TF: FNR [P0A9E5, view regulon]
Reported TF sp.: Escherichia coli str. K-12 substr. MG1655
Reported site sp.: Escherichia coli str. K-12 substr. MG1655
Created by: Sepideh Zahiri
Curation notes: -

Experimental Process

Linear DNA carrying the resistance cassette and flanking regions was generated by PCR.β-galactosidase assays approached to measure the B-galactosidase activity after overnight incubation of the broth containing appropriate antibiotics in anaerobic conditions at 37 °C.site-directed mutagenesis of the FNR site was achieved using PCR and appropriate synthetic oligonucleotides. Footprinting reactions with FNR were done essentially with PydhY used as the target DNA

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TTGATAACGATCAA

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence	Regulated genes	Gene diagram	Experimental techniques	TF function	TF type
TTGATAACGATCAA	ydhY,		Experimental technique details DNAse footprinting (ECO:0005631) DNAse footprinting - ECO:0005631 The DNAse foot-printing method starts by focusing on a given region of interest (e.g. a promoter region) and amplifying it by PCR to obtain lots of sample. It then throws in the TF and then the DNAse. The mix is left to stir for a short time and then gel electrophoresis is run to compare the pattern of fragments in a control (no TF) and in the sample. If the TF has bound the sample, it will have protected a stretch of DNA (encompassing some fragments of the control) and thus those fragments will not appear in the sample gel. The fragments can then be cut-out from the gel, purified and sequenced to obtain the sequence of the protected region. This is often used to identify the binding motif of a TF for the first time. The foot-printing will typically resolve the protected region down to 50-100 bp, and the sequence can be then examined for possible TF-binding sites either by eye of using a computer search. - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) qPCR [quantitative real-time] - ECO:0005660 Quantitative PCR (or quantitative real time polymerase chain reaction) is a modification of the conventional PCR reaction used to amplify and simultaneously quantify a targeted DNA molecule. In its more standard incarnation, a inactive fluorescent dye is added to the PCR mix. This dye is activated upon binding double-stranded DNA; as the PCR iterates, there is more and more dsDNA and therefore more fluorescence intensity. -	activator	dimer