(B) The DNA-binding assays for MtrA on different DNA substrates. The EMSA reactions (10 μl) for measuring the mobility shift contained 200 fmol 32P-labeled DNA and increasing amounts of MtrA proteins (100 nM-600 nM). The protein/DNA complex is indicated by arrows on the right of the panels. (C) Schematic representation of conserved motifs

located downstream of two dnaA promoters. The base-pair numbers far from the start codon of the dnaA gene are indicated. selleck screening library The interaction between MtrA and these two sequence boxes was further confirmed by DNase I footprinting assays (Fig. 3). Regions that contain these two boxes were significantly protected when MtrA was present. Protection at S6 occurred at all MtrA concentrations while the protection of S7 was dependent on the concentration of MtrA. This suggests that MtrA has different binding affinities with these regions. Figure 3 MtrA footprinting analysis in the M. tuberculosis dnaA HMPL-504 promoter PLX3397 purchase region. (A) DNase I footprinting assay of the

protection of two short dnaA promoter regions (S6 and S7) against DNase I digestion by MtrA. The substrate S6 contains S1 and S2 sequences, and the substrate S7 contains S5 sequences. The ladders are shown in the right panel and the obtained nucleotide sequences are listed. The protected regions are indicated. The two specific sequence boxes are indicated by “”*”". (B) Summary of MtrA footprinting analysis in the M. tuberculosis dnaA promoter Molecular motor region. The DNA sequence correspond with

the dnaA promoter region from -303 to -1. The position of two transcription start sites (P1dnaA and P2dnaA), two footprint regions, and two MtrA binding boxes are indicated. We characterized two sequence boxes for the recognition of MtrA within the dnaA promoter, situated immediately downstream of promoters P1 and P2. The binding sequence boxes and their situation within the dnaA promoter are summarized in Fig. 2C. Characterization of potential target genes regulated by MtrA in mycobacterial genomes We searched the intergenic regions of the M. tuberculosis and M. smegmatis genomes extensively based on the two sequence motifs for MtrA in the dnaA gene promoter region. To validate the target genes, several regulatory regions of the genes were amplified. The DNA-binding activities of MtrA were examined using EMSA assays. As shown in Fig. 4, the regulatory sequence of a predicted target gene, isoniazid inducible gene iniB (rv0341), could be recognized by MtrA. A specific DNA/protein complex band was also observed. In addition, MtrA was able to bind with two target promoter DNA sequences of Rv0574 (a hypothetical protein) and Rv3476 (KgtP), producing a corresponding DNA/protein band (Fig. 4A). The positive target DNA was shown to bind with MtrA, while the negative DNA was not. The 7 bp sequence motif could also be found in the promoter regions of two previously characterized target genes, CgmepA and CgproP, in C. glutamicum. Interestingly, M.