Figure 6
RhaR binds to the promoter region of rhaA. (A) SDS-PAGE analysis of the purification of the His6::RhaR (1-159*) protein expressed in E. coli. Lysates from uninduced and IPTG induced cells transformed with the empty plasmid (lanes 1 and 2, respectively) and the plasmid bearing the RhaR expression cassette (lanes 3 and 4, respectively). Lanes 5 and 6: Soluble fraction of the lysate before and after passage through the affinity column. Lanes 7-9: 300 mM imidazol elution fractions. The predicted molecular mass of the protein is 19,9 kDa and its position its arrowed. (B) Mobility shift assays of rhaR p and xlnA p (negative control promoter). The mobility of each PCR fragment (100 ng) was assayed in the presence of increasing amounts of His6::RhaR (1-159*) protein (from 0 to 1.2 μg). (C) Identification and delimitation of a potential binding site for RhaR in rhaA p. rhaA p was divided into 4 overlapping fragments the sizes and locations of which are indicated in the upper part of the panel. Each restriction fragment was assayed with 0 and 240 ng of the protein. (D) Effect of point mutations on the proposed RhaR binding site. EMSAs using wild type and mutated fragments or double stranded oligonucleotides containing the predicted RhaR site. Fp indicates the mobility of the free probes. Arrows show the positions of the RhaR-DNA complexes.