Identification of the DNA-binding preferences of Ste12 and Tec1 by HT-SELEX. Identification of the DNA-binding preferences of Ste12 and Tec1 by HT-SELEX. (A) Heatmap showing relative frequencies of the possible orientations and spacings of the primary 6-mer selected in Ste12 (TGAAAC, Top) and Tec1 (GAATGT, Middle) binding reactions. The Bottom heatmap shows the frequency of each respective 6-mer in the cobinding sample containing both proteins. In the cobinding sample, we excluded sequences with homodimeric Ste12 sites, which were present at a 30-fold higher level than heterodimeric sites. The single dark green box in the Top and Bottom heatmaps indicates the most frequent orientation and spacing of sites; white boxes are at most 30% as frequent as the maximum. No frequent dimeric site organizations were observed for Tec1 (Middle). (B) Full motifs identified by Autoseed software (1). (C) Instances of the Ste12 homodimeric motif, with sites oriented tail-to-tail with a 3-bp spacer, were used to query native yeast promoters. Two pheromone-induced genes, GPA1 and STE12, contain two perfect sites, while most other genes contain a perfect site paired with a site containing one or two mismatches, with the 3-bp spacing intact. (D) A similar search of yeast promoters using the preferred Ste12 and Tec1 heterodimeric site identified a set of invasion-associated genes, as well as those not previously linked to invasion. Michael W. Dorrity et al. PNAS 2018;115:34:E7997-E8006 ©2018 by National Academy of Sciences