FIG. 5.
LuxR-3OC6-HSL stimulation of transcription from the luxI promoter. A 612-bp double-stranded linear DNA fragment carrying the luxI-luxR promoter region was used as a template in an in vitro transcription runoff assay. Reaction mixtures contained 1 nM DNA template, 10 nM E. coli RNA polymerase, [α32-P]ATP-labeled deoxynucleoside triphosphates, and 10 μM 3OC6-HSL, either with (lanes 1 to 6) or without (lane 7) purified LuxR, as indicated. Open arrows indicate calculated positions of the 395- and 130-base molecular size standards. Filled arrow points to the 300-base luxI runoff transcript. Because no significant transcription is seen in the absence of LuxR (lane 7), the LuxR-dependent transcripts above the 300-base luxI band are likely due to transcription that initiated at the luxI promoter but was extended beyond the template 3′ end by a loop-around mechanism described previously (24).