Fig. 3: FruR-DNA recognition pattern is altered upon F1P binding.

A Potential FruR-binding sites are depicted with positions relative to the TSS of the fru promoter. The bent arrow indicates the TSS, and each 10 bp binding motif is represented by a red arrow, indicating the 5’-3’ direction of the repetitive sequence. Bases identical to the TGAWWGWTTT consensus sequence are shown in red in each motif. B Mutant DNA fragments (−218 to +71 relative to the TSS; 40 ng), where the indicated motif was replaced by CATTTTCGCC, were incubated with FruR (1 μg), and their binding affinities were compared by EMSA. C The effects of HPr2 and F1P on FruR’s DNA binding pattern were assessed. Each DNA fragment (40 ng) was incubated with FruR (2 μg), HPr2(S46D) (1 μg), and F1P (0.5 mM). EMSAs were conducted on a 6% TBE polyacrylamide gel. D The effects of HPr2, F1P and FruR on RNAP binding were evaluated by DNase I footprinting. A 6-FAM-labeled reverse-complemented DNA probe (200 ng) covering −318 to +171 relative to the TSS of the fru promoter was incubated with E. coli RNAP holoenzyme (1 μg), FruR (1 μg), or both in the presence of either F1P (1 mM), HPr2 (1 μg) or HPr2(S46D) (1 μg) as indicated, prior to digestion with DNase I. Each motif is labeled with black arrows indicating the 5’-3’ direction above the panel. The motifs protected by FruR and RNAP binding are labeled with red and blue arrows, respectively, below the panel. RNAP protection sites are underlined in blue. The fluorescence intensity of the 6-FAM-labeled fragments is shown on the y-axis of each electropherogram and fragment sizes were determined by comparison with the internal molecular weight standards. E In vitro transcription assays were conducted using DNA templates (1 μg) with mutations in one motif of the fru promoter (“mut#”). Each DNA template was mixed with E. coli RNAP holoenzyme (1 μg), FruR (4 μg), F1P (1 mM), and HPr2(S46D) (4 μg). RNA products were purified, annealed with a HEX-labeled primer, and subjected to cDNA synthesis as described in Fig. 2A. Transcription levels were determined by measuring the area under the peak of the fruR transcript in arbitrary fluorescent units, relative to the transcription level of the wild-type template (WT). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, n.d.: not detected). Data are presented as means and SD (n  =  3, independent measurements).