Figure 6: Interaction of GrlA with the ler regulatory region in vitro.
From: Structure of GrlR–GrlA complex that prevents GrlA activation of virulence genes
(a) EMSA assay for PCR DNA fragments comprising the ler regulatory region (ler−275/+217; numbers indicate the number of base pairs upstream and downstream from the functional ATG start codon, respectively) DNA VBP is a non-specific DNA. (b) EMSA assay with substituted MBP-GrlA proteins. PCR DNA fragments comprising the regulatory region (ler−275/+217) were mixed and incubated with 1.0 μM of purified MBP-GrlA, or with different substituted mutants. Free DNA and protein–DNA complexes were resolved by 5% PAGE and stained with ethidium bromide. The GrlA species used include: WT GrlA, GrlA R53A, GrlA R54A, GrlA R64A, GrlA R65A, GrlA K66A, GrlA Y78A, GrlAR65AK66A and ler pr (ler −275/217). (c) Competitive EMSA assay aimed at testing competition between DNA and 6His-GrlR for binding to MBP-GrlA. PCR DNA fragments comprising the regulatory region (ler−275/+217) were mixed and incubated with 1 μM purified MBP-GrlA for 15 min, then combined with increasing concentrations (0.0, 0.15, 0.3, 0.45 and 0.6 μM) of 6His-GrlR for an additional 15 min. The complexes and free DNA were separated on a 5% native polyacrylamide gel and stained with ethidium bromide (ler pr: ler −275/+217).
