Figure 2
Identification of the GabR-binding site in the gabT promoter. Panel A, SDS-PAGE analysis of GabR (57 kDa) expressed in E. coli (pET21b-gabR) and purified by nickel affinity column chromatography. M, protein marker. Panel B, Electrophoresis mobility shift assay (EMSA) of the gabT promoter fragment (286 bp) after interaction with GabR. Lane 1, FAM-labeled PgabT probe; lanes 2–8, incubation of the probe with increasing concentrations of purified GabR indicated at the top of the figure. Each lane contained 0.1 μg of probe. Lane 9, incubation of a five-fold greater amount of unlabeled PgabT probe mixed with labeled PgabT probe and 8 μM GabR. Panel C, protection of a 96-bp sequence in the gabT promoter by GabR, as revealed in a DNase I footprinting protection assay. The fluorograms correspond to the control DNA (10 μM BSA) and to the protection reactions (with 3.2, 4.8 and 8 μM of GabR). Panel D, β-galactosidase activity assay of the gabT promoter with the GabR-binding site (●), without the 60-bp GabR-binding site (▲), and without the first 21-bp repeat region of the GabR-binding site (◊). The deletion sequence is that underlined in Figure 2C. T0 is the end of exponential phase, and Tn is n hours after T0.