Figure 2
Determination of the PmlR2-binding sites by DNase I footprint analysis. (A) The nucleotide sequence of P6650. PmlR2-binding sites are underlined. The consensus (5′-T(G/A)TACA and TGTACA) for P. putida PplR1-binding is denoted by bold letters including Site 1 and Site 2. The transcriptional start site of P6650, as determined by 5′-RACE and CAGE, is indicated by + 1 and a vent arrow. Putative − 10 and − 35 hexamer sequences are shown in small letters. (B) DNase I footprint assay. One PmlR2-binding site in the sense ( +) and antisense ( −) strands of P6650 was protected. The amounts of recombinant PmlR2 used were 0 pmol (lane 1 and 6), 10 pmol (lane 2), 20 pmol (lane 3), 40 pmol (lane 4), and 80 pmol (lane 5), which were added to the reaction mixture with 10 kcpm 32P-labeled DNA probes. Three independent experiments in technical replicates were performed, and representative data were shown. The original and unprocessed versions of the full-length gels were included in Figs. S10 and S11. (C) The alignment of the two PmlR2-binding-sites with the P. putida PplR1 consensus.
