Curation Information

Publication: Mutational analysis of the bglH catabolite-responsive element (cre) in Lactobacillus plantarum.;Marasco R, Muscariello L, Rigano M, Sacco M;FEMS microbiology letters 2002 Feb 19; 208(1):143-6 [11934508]
TF: CcpA [F9UQH6, view regulon]
Reported TF sp.: Lactobacillus plantarum LM3
Reported site sp.: Lactobacillus plantarum LM3
Created by: Dinara Sagitova
Curation notes: -

Experimental Process

EMSA confirmed binding of CcpA to the bglH promoter region. Introduction of a single substitution in the CcpA binding site drastically lowered the affinity of cre–protein binding in vitro. Comparison of the expression level of the wild type and mutated bglH promoter with the cat reporter gene confirmed CcpA-mediated repression of bglH.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TGTAAGGGCTATCA

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
TGTAAGGGCTATCA	pbg9		Experimental technique details CAT reporter gene assays (ECO:0005617) CAT reporter gene assays - ECO:0005617 A reporter assay based on the fusion of a promoter of interest with the cat gene, which produces chloramphenicol acetyltransferase, capable of being acetylated by acetyl CoA. The amount of acetylated chloramphenicol is directly proportional to the amount of CAT enzyme present. - 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 Site directed mutagenesis (ECO:0005667) Site directed mutagenesis - ECO:0005667 Target-specific mutation, as opposed to non-specific mutation. In the context of TF-binding sites, site-directed mutagenesis is typically used to establish/confirm the specific sequence and location of a site, often in tandem with EMSA. Different positions of a putative binding site are mutated to non-consensus (or random) bases and binding to the mutated site is evaluated through EMSA or other means. Often implemented only in conserved motif positions or serially through all positions of a site. -	repressor	not specified