Figure 3. Regulation of the hctB promoter by EUO.

A. The hctB promoter is repressed at a higher EUO:DNA ratio. Graph showing the effect of 2.5 µM rEUO on transcription of the hctB promoter at a plasmid DNA concentration of 13 nM (gray bars) or 3 nM (white bars). The hctB promoter was transcribed by σ²⁸ RNA polymerase, while σ⁶⁶-dependent control promoters (omcAB and dnaK P1) were transcribed with E. coli holoenzyme. For each promoter, transcription in the presence of EUO was normalized to baseline transcription in the absence of EUO. Values are from the average of at least three independent experiments with standard deviations indicated by error bars. B. DNA sequence of the C. trachomatis hctB promoter, with the putative 15 bp core EUO-binding site indicated with a double underline, and the C residue at position −12 marked with a carat. The −35 and −10 promoter elements are labeled. C. Representative gels showing in vitro transcription of the wild type (WT) hctB promoter and mutant promoters containing a single nucleotide substitution of the C at position −12. Transcriptions were performed with σ²⁸ RNA polymerase in the absence or presence of 2.5 µM rEUO. Only 1/4 of the transcription reactions with the WT promoter were loaded on the gel because this promoter was transcribed at much higher levels than the mutant promoters. D. Graph showing quantification of these transcription results. Transcript levels from the WT promoter were defined as 100%, and transcript levels from each mutant hctB promoter were normalized to the WT promoter. E. Graph showing effect of EUO on transcription of WT and mutant hctB promoters. For each promoter, transcription in the presence of EUO was normalized to baseline transcription in the absence of EUO. Values are from the average of at least three independent experiments with standard deviation indicated by the error bar.