Figure 2.

Determination of NF-CO Sequence Specificity in Vitro.

(A) CORE2 competitors and mutagenesis strategy. The unlabeled wild-type CORE2 and 30-mer oligo sequences with mutated nucleotides are shown, as in Figure 1, for the three sets of CORE2 mutant oligos (mI, mII, and mIII). In the bar graphs, mI and mII mutant oligo competitor efficiency (competition) is expressed as ratio of the dose-response curve slope of the mutant versus the wild-type oligo (see Methods). Competition of the wild-type oligo is set as 1. Indicated values represent the mean of three (mI oligos; top panel) or two (mII oligos; bottom panel) series of competition assay experiments (see also Supplemental Figure 4). Error bars indicate ± sd for mI oligos or value ranges for mII oligos. Sequences in red boxes highlight mutations with reduced competition (<0.67 of wild-type oligo efficiency).

(B) and (C) CORE2 mIII mutant oligo EMSA competition results. Competition efficiencies are shown as mean value of three independent series of experiments for each of the mIII single nucleotide mutant oligo, as indicated for (A). For each mutated position, the wild-type oligonucleotide competition value, set as 1, is also shown. Values are also displayed in the bar graph in (C) (average of three independent sets of experiments ± sd). For each nucleotide position, dark and light shaded bars denote mutant and wild-type (asterisk) competitors, respectively (see also Supplemental Figure 4). In (C), the sequence matrix obtained with the mIII competitions (information content) is shown on the right, for the sense (+) and reverse (−) strands of the FT promoter.