A Novel Branched Chain Amino Acids Responsive Transcriptional Regulator, BCARR, Negatively Acts on the Proteolytic System in Lactobacillus helveticus
Figure 5
Electrophoresis mobility shift assay with various DNA fragments of the region upstream of the pepV.
Mobility shift assay with or without 2.3 µM BCARR protein and 10 mM BCAAs (A). Each 2,500 cpm of 32P labeled DNA fragment (0.3 ng) was incubated as described in Materials and Methods. Protein-DNA complexes were analyzed by 10% polyacrylamide gel electrophoresis. The 32P signals in the gel were detected by exposure to X-ray film. The position of the shifted bands is indicated in the right margin by arrows (weak shift indicated by a small arrow, and a significant shift by a big arrow; the origin is indicated by the open arrow). DNA fragments used in the above experiment and the results of the EMSA are summarized in (B). Nucleotide positions are relative to the start codon. BCARR-Box indicates the predicted BCARR binding motif by MEME analysis. ORF shows the open reading frame of the pepV gene. The intensity of the shifted bands is indicated by + (slight probe shifted) to +++ (all probes shifted).