Fig. 4

EMSA of the PcRsmA binding activity in the pcbAB-pcbC intergenic region. A The pcbAB-pcbC intergenic region is shown at the top, with the position of the six probes used for screening the DNA binding activity of PcYap1 (Fig. 2D) and PcRsmA. The position of the DNA binding sites of PcYap1 and PcRsmA is shown with a bar of red and violet color, respectively. The small lines above or below the bars indicate the position of the binding sequence in the upper or lower strand, respectively. Below it is shown the relative position of seven probes located within the DNA region corresponding to probe 5, with the sequence of the upper strand from the smaller probes at their right. B Screening of the entire pcbAB-pcbC intergenic region to locate target sites of PcRsmA; lanes 1 through 6 correspond to binding reactions between probes 1 through 6 with PcRsmA. At the right are shown confirmation binding reactions of PcYap1 and PcRsmA with probe 5, and a positive control reaction of PcRsmA with probe AflR-RsmA of 25 bp (Additional file 12), which belongs to a region in the promoter of the A. nidulans sterigmatocystin cluster gene aflR containing a TGACACA sequence proven to be bound by A. nidulans RsmA [44]. C Positive binding reaction of PcRsmA with the probe upPta1 and negative reaction with the probe dwPta1. D Location of the PcRsmA binding sequence; the probes containing the sequence TGAGACA (RsmA-2 and RsmA-2C) are bound by PcRsmA, whereas the probes lacking this sequence (RsmA-2A and RsmA-2B) are not. E Specificity test for the binding of PcRsmA to the TGAGACA sequence. We used the probe RsmA-2C-M1, containing the mutated sequence TGCGATA, to prove the specificity. RsmA-2C* and RsmA-2C-M1* are labelled probes; RsmA-2C and RsmA-2C-M1 are cold (unlabelled) probes, which were added in excess amounts of 50x, 100x, 150× and 200× as indicated at the top of the gel image