FIGURE 2.
HrtR controls hrtRBA transcription via binding to a 15-nt palindromic sequence in the hrtRBA promoter. A, HrtR is a transcriptional repressor of the hrtRBA operon. Transcriptional fusions between the hrtRBA promoter region, including or not the coding region of HrtR (Phrt-lac and PhrthrtR-lac, respectively), were expressed in the ΔhrtRBA L. lactis strain. β-Gal activity was quantified by luminescence (see “Experimental Procedures”; results shown for at least three experiments) B, HrtR binds the promoter region of hrtRBA. EMSA shows specific binding of HrtR to the hrtRBA promoter. 30 pmol of the hrtRBA promoter fragment (lanes 1 and 2) and of the hrtR-coding region (lanes 3 and 4) were incubated in the presence of 200 pmol (lanes 2 and 4) of MBP-HrtR. C, a 15-nucleotide palindromic motif (in boldface type and boxed; the central adenine is in boldface type) is present upstream of hrtRBA. The start codon is in gray, and the RBS sequence is underlined. D, the palindrome is needed for HrtR-DNA binding. EMSA was done using a native DNA fragment encompassing the promoter region, or a fragment in which the 15-nt palindromic sequence was substituted with a random sequence. A gel representative of three experiments is shown. RLU, relative light units.