GlnR Binding to nif Operon Promoter

EMSAs were performed as described previously using a DIG Gel Shift Kit (2nd Generation; Roche, USA) [12 (link)]. The promoter fragments of nif operon were synthesized by Sangon Biotech Co., Ltd (Shanghai). Two DNA fragments corresponding to the sequences of the first strand and the complementary DNA strand were synthesized. The two strands were annealed and then labeled at the 30 end with digoxigenin (DIG) using terminal transferase, and used as probes in EMSAs. Each binding reaction (20 μl) consisted of 1 μg poly [d(A-T)], 0.3 nM labelled probe, and various concentrations of purified His6-GlnR in the binding buffer. Reaction mixtures were incubated for 30 min at 25°C, analyzed by electrophoresis using native 5% polyacrylamide gel run at 4°C with 0.5×TBE as running buffer, and electrophoretically transferred to a positively charged nylon membrane (GE healthcare, UK). Labelled DNAs were detected by chemiluminescence according to the manufacturer’s instructions, and recorded on X-ray film. The primers used here are listed in S6 Table.