A Novel marRAB Operon Contributes to the Rifampicin Resistance in Mycobacterium smegmatis
Figure 4
Identifying the DNA-binding motif of MarR.
(A) DNaseI footprinting assays were carried out on the coding and non-coding strands. Protection of the promoter DNA by MarR against DNaseI digestion was tested by increasing the amount of MarR (0–0.6 µM). The ladders are shown and the corresponding nucleotide sequence is listed (lanes 2–4). The protected regions on the coding and non-coding strands are indicated. (B) Sequence and structural characteristics of the promoter DNA region protected by MarR. The regions protected by MarR are underlined. The 21-bp sequences containing the inverted repeats (IR) separated by 1 bp are indicated by a pair of arrows. The translation start codon of MarR is indicated in bold. (C) EMSA assays for the DNA-binding activity of MarR on DNA substrates with wildtype IR sequence and IR-deleted mutant sequences. DNA substrates were co-incubated with 0.2–0.6 µM of the MarR protein. Cold DNA substrate containing IR motif (p4), but not unrelated substrate (p3) which does not contain IR motif, could competitively inhibit the binding of MarR to the labeled DNA substrate (p4*).
