Using the primer extension analysis, the promoter region of tpx gene was identified. The sequence with a high degree of homology (18/26) to the E. coli IscR consensus binding site was identified at positions -46 to -21 of the tpx promoter. tpx expression was monitored with a WT strain and a iscR knockout mutant. "Northern blot analysis was performed using RNA samples prepared from uninduced and PB-induced iscR mutants, in addition to its complemented strain harboring pIscR plasmid (iscR/pIscR) and a PAO1 strain. As expected, PB-treated PAO1 showed a 6.5-fold induction in tpx expression based on densitometric analysis. The PB induction of tpx expression was abolished in the iscR mutant. The tpx expression showed constitutively high expression levels in the iscR mutant. The uninduced level of tpx transcripts in the iscR mutant was 10.5-fold higher than the uninduced level in PAO1 and 1.4-fold higher than the levels attained in the PB-induced sample from PAO1. In the iscR/pIscR strain, the uninduced tpx transcript level was 0.7-fold lower than the uninduced level in the PAO1 strain. As expected, the exposure to PB induced a 2.5-fold increase in the tpx transcription level. PB-induced tpx expression was restored in the complemented mutant strain." A gel mobility shift assay confirmed specific binding of IscR to the tpx promoter region.
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|