Figure 5.

The PorX response regulator binds to the porT promoter region in vivo and in vitro.A, mapping transcription start site of the porT gene in 33277 strains. Primer extension products from wild-type (WT, Lane 2 in the figure) and ΔporX (YS19145, Lane 3 in the figure) strains were generated using primer 4025 and total RNAs isolated from wild-type strain and ΔporX mutant (YS19181) grown in BHI medium for 48 h. M (Lane 1 in the figure) corresponds to a DNA ladder derived from a Maxam–Gilbert A + G reaction from a DNA fragment amplified with primers ³²P-3043 & 3044. Arrow indicates the transcription start (A, also labeled as +1, also see next) representing 90th nucleotide from the 3’ end of the ladder. B, the DNA sequence of the porT promoter region. Underline corresponds to the PorX-protected regions (I and II) characterized below. Underlined capital letters represent the conserved nucleotides in both PorX-binding sites. Numbering begins from the transcription start site (+1) determined above and labeled with an arrow. C, DNase I footprinting analysis of the porT promoter fragment amplified with primers ³²P-4025 & 4026 for the coding strand and increasing amounts of PorX-c-His₆ protein (0, 70, 140, and 280 pmol, respectively, see Lanes 2–5 in the figure). Products were separated in polyacrylamide DNA sequencing electrophoresis, and the bands were detected by autoradiography. Right lines correspond to the PorX-protected DNA sequences and are labeled as I and II, respectively. The ladder M (Lane 1 in the figure) corresponds to the same ³²P-labeled porT promoter fragment and degraded by the Maxam and Gilbert reaction.