Crystallization and data collection

Fis protein and DNA Fis, under the control of the T7 RNA polymerase (Pan et al. 1996), was expressed by auto-induction (Studier 2005). Extracts were prepared using a French Press and nucleic acids were removed by precipitation with 0.35% polyethyleneimine followed by precipitation with ammonium sulfate (80% saturation). Fis was purified by FPLC chromatography over a Bioscale S20 (Bio-Rad) column, followed by Superdex 75 gel filtration (GE Healthcare) performed in 50 mM HEPES (pH 7.5), 1 M NaCl, 1 mM DTT, and 1 mM EDTA. Fis-N84A was created using the QuikChange method. Oligonucleotides (IDT) for crystallography were annealed by slow cooling in 20 mM TrisHCl (pH 8.0) and 150 mM Na acetate at a final duplex concentration of 25 mg/ml. For DNA binding assays the top strand of the duplex was 5’-P-labeled using T4 polynucleotide kinase and annealed with 1.5 molar excess of the bottom strand. The radiolabeled duplex probes were purified by PAGE. The EMSA in Fig. 1A utilized a 97 bp EcoR1-HindIII fragment from pUC19 containing the F1 sequence (31 bp) cloned into the Sma I site (pRJ2484).