Figure 8
Point mutation analysis of the HpaR1 binding sequence in gumB promoter.
(A) Six nucleotides were randomly chosen to be mutated and the resulting mutant fragments were designated as M1-M6. (B) FAM-labelled 287-bp gumB promoter DNA fragments (1.0 nM) with variant point mutations (M1-M6) or without a mutation (wild type) were incubated with increasing amounts of His6-HpaR1 protein (0, 5, 10, 15 and 20 nM) for 20 min at 30 °C before the electrophoretic mobility shift assay. The activity of these mutant promoters was detected by constructing promoter-gusA transcriptional fusion reporter plasmids and comparing their GUS activities in wild-type and hpaR1 mutant backgrounds. b, bound DNA; f, free DNA.
