Curation Information

Publication: Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus.;Boyd CH, Gober JW;Journal of bacteriology 2001 Jan; 183(2):725-35 [11133968]
TF: CtrA [P0CAW8, view regulon]
Reported TF sp.: Caulobacter crescentus NA1000
Reported site sp.: Caulobacter crescentus NA1000
Created by: Dinara Sagitova
Curation notes: -

Experimental Process

flgB transcription start site was determined by S1 nuclease protection assays. Visual sequence inspection of the flgB promoter identified the presence of a consensus CtrA binding sequence (TTAA-N7-TTAA). DNase I footprinting confirmed that CtrA interacts with the flgB promoter by binding to a 28-bp region. Comparison of the expression levels by beta-gal reporter assays in the wild-type and ctrA temperature-sensitive C. crescentus strains, indicated that CtrA represses flgB expression and activates fliO-fliP expression. Additionally, B-Galactosidase activity was measured from the promoters containing mutations within the consensus CtrA binding sequence and outside of it. The authors report that the results of this analysis were complex. Among the results obtained from these site-directed mutagenesis experiments, it was shown that mutation in the conserved TTAA to GCAA had a deleterious effect on both flgB and fliO-fliP expression.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TTAATGCCGGATTAA

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
TTAATGCCGGATTAA	fliO, fliP, flgB,		Experimental technique details Beta-gal reporter assay Beta-gal reporter assay Reporter assay using the beta-galactosidase (lacZ) gene. The lacZ gene is typically fused to the promoter of interest. Differential regulation of the promoter mediated by the TF is assessed by induction of the system and evaluation of lacZ expression. Bacteria expressing lacZ appear blue when grown on a X-gal medium. The assay is often performed using a plasmid borne construction on a lacZ(def) strain. - 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 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. - Experimental technique details Visual sequence inspection (nan) Visual sequence inspection - nan Visual inspection of promoter region to identify putative binding sites based on previous knowledge -	dual	not specified