Curation Information

Publication: The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions.;Shimada T, Ishihama A, Busby SJ, Grainger DC;Nucleic acids research 2008 Jul; 36(12):3950-5 [18515344]
TF: RutR [P0ACU2, view regulon]
Reported TF sp.: Escherichia coli BW25113
Reported site sp.: Escherichia coli BW25113
Created by: Erill Lab
Curation notes: -

Experimental Process

ChIP-chip to detect sites. Motif discovery and site search to identify sites. EMSA to validate targets. Northern blot to validate regulation.

ChIP assay conditions: For experiments with exponentially growing cells, overnight cultures of E. coli strain BW25113 or JW0998 were diluted 1:100 into fresh medium either with or without uracil, and grown for ∼4 hours to an OD650 of 0.3–0.4.
ChIP notes: Assays were done either in triplicate (−uracil) or in duplicate (+uracil). Briefly, cultures of E. coli BW25113 and, as a control, JW0998 ΔrutR were grown to mid-log phase at 37°C. Cells were then treated with 1% formaldehyde and broken open by sonication which also fragments cross-linked nucleoprotein. Cross-linked RutR–DNA complexes were immunoprecipiated from cleared lysates of BW25113 using anti-RutR rabbit polyclonal anti-serum, and parallel samples were isolated from control JW0998 ΔrutR cells. Cross-links were then reversed and immunoprecipitated DNA was purified. DNA samples isolated from BW25113 cells and the control ΔrutR cells were labelled with Cy5 and Cy3, respectively. To identify segments of DNA specifically associated with RutR, the two labelled samples were combined and hybridised to a 22 000 feature DNA microarray (Oxford Gene Technology, Oxford, UK). For each probe, the Cy5/Cy3 ratio was measured and this was plotted against the corresponding position on the E. coli BW25113 chromosome, creating a profile of RutR binding (Figure 1). We then selected ‘peaks’, formed by two or more consecutive probes, with a Cy5/Cy3 ratio of >2.5. To increase the stringency of our search, we discarded the small number of peaks where the RutR-binding signal was not reduced at least two-fold when uracil was added to cultures. The centre of each peak is defined as the centre of the probe within the peak that had the highest Cy5/Cy3 signal.

Transcription Factor Binding Sites

TTTACCACCTGGTCCG

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
TTTACCACCTGGTCCG	ves,		Experimental technique details ChIP-chip (ECO:0006007) ChIP-chip - ECO:0006007 The principle of ChIP-chip is simple. The first step is to cross-link the protein-DNA complex. This is done using a fixating agent, such as formaldehyde. The cross-linking can later be reversed with heat. Cross-linking kills the cell, giving a snapshot of the bound TF at a given time. The cell is then lysed, the DNA sheared by sonication and the chromatin[2] (TF-DNA complexes) is pulled down using an antibody (i.e. immunoprecipitated). If an antibody for the TF is available, then it is used; otherwise, the TF is tagged with an epitope targeted by commercially available antibodies (the latter option is cheaper, but runs the risk of altering the TF's functionality). Cross-linking is then reversed to free the bound DNA, which is then amplified, labeled with a fluorophore and dumped onto a DNA-array. The scanned array reveals the genomic regions bound by the TF. The resolution is around ~500 bp as a result of the sonication step. - Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details Northern blot (ECO:0005653) Northern blot - ECO:0005653 The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. -	repressor	dimer