The G-SELEX method, purification and DNA binding of Gcn4. (A) Overall schematic of G-SELEX method: Purified genomic DNA is sonicated, ligated to the Illumina paired-end adaptor (black lines capped with black squares), and amplified by PCR. An excess of DNA is incubated with Gcn4 (green semicircle) bound to Ni-NTA beads (gray octagon) at 4°C overnight. Gcn4-bound DNA is eluted from the beads, purified, amplified, and used for a second round of Gcn4 binding. After three rounds, the eluted DNA is indexed and sequenced. (B) Purification of recombinant Gcn4. Purified Gcn4 (0.3 μL and 3 μL) analyzed by SDS-PAGE. The major band accounts for ∼50% of the protein (by densitometry). Gcn4 containing a 6xHis tag binds tightly to the Ni-NTA magnetic beads used for the experiments (lane “B”: loaded beads). (C) Gcn4 binds to a fluorescently labeled, double-stranded 24-mer containing the AP-1 site located in the ARG1 promoter (EMSA). DNA (10 nM) was incubated for 30 min at room temperature with 0, 2.5, 5, 10, 20, 40, 80, 160, 320, 1000 nM Gcn4 and 500 ng of unlabeled poly(dI/dC) (left) or poly(dA/dT) (right) before loading in a polyacrylamide gel.