Extended Data Fig. 1: Purification of GAF-DBD protein.
From: GAGA zinc finger transcription factor searches chromatin by 1D–3D facilitated diffusion
a, Subtractive Ni-NTA purification after “one-pot” reaction which cleaved off 6xHis-SUMO and labeled the N-terminus of GAF-DBD with Cy3. The calculated molecular weights are 34.4 kDa for 6xHis-SUMO-Cys-GAF-DBD, 21.0 kDa for Cys-GAF-DBD and 13.4 kDa for 6xHis-SUMO. This experiment was independently repeated once with similar results. b, EMSA showing GAF-DBD binds to hsp70 promoter NCP DNA with K1/2 ~ 12 nM. GAF-DBD concentrations are, from left to right, 0, 4, 8, 12, 16, 20, 24, 28, 32 nM and the DNA concentration is 3 nM. This experiment was independently repeated once with similar results. c, The binding frequency of GAF-DBD on cognate DNA as a function of protein concentration (For each concentration, N = 40 traces from two imaging sessions were used to quantify the binding frequency). The binding constant is determined from a linear fit to the data (R2 = 0.99) to be 0.27 s−1 nM−1. To calculate KD, we divide the measured kon (3.1 s−1, Fig. 1) by this number to get 11.5 nM which agrees well with the 12 nM apparent KD (K1/2) determined from the EMSA in b. Data are represented as mean values +/− SD. d, Schematics of GAF-FL short isoform, GAF-DBD and NURF-binding regions69 to scale. e, Native hsp70 promoter DNA sequence. GAF cognate sites are highlighted in red. TATA box in bold.
