Figure 3
From: The mechanism of a formaldehyde-sensing transcriptional regulator
Formaldehyde enhances disassociation of the Pfrm-EcFrmR complex.
(a) Bio-Layer Interferometry (BLItz) assays. Reactions to evaluate the interaction of biotin-labeled Pfrm DNA, immobilized on a streptavidin probe, with EcFrmR were carried out with 10 different concentrations of EcFrmR (Table S4A). Representative traces for EcFrmR (6.16 μM tetramer, black line, 0.88 μM tetramer; red line), as well as EcFrmR pre-treated with 200-fold molar excess of formaldehyde (0.88 μM tetramer; blue line), and EcFrmR binding at a non-target DNA (PydhY, 0.88 μM EcFrmR tetramer; green line) are shown. (b) Pre-formed Pfrm-EcFrmR complexes were exposed to 10 different concentrations (Table S4C) of formaldehyde and disassociation curves were recorded. Traces for 0 (black); 0.05 mM (orange); 0.25 mM (gray); 0.62 mM (yellow); 1.25 mM (blue); 3.69 mM (green); 4.92 mM (dark blue); 7.38 mM (brown) are shown. (c) Single exponential fits to formaldehyde disassociation curves were used to obtain the observed rate constants (kobs) which were plotted against formaldehyde concentration to obtain the apparent second order rate constant. (d) Inhibition of frmRAB transcription by EcFrmR in vitro is relieved by formaldehyde. Reaction conditions are described in the Methods section. Left panel, Pfrm; right panel, Pndh. Lanes 1, RNA size markers, top to bottom: 600, 500, 400, 300, 200, 100 bases; Lanes 2, no EcFrmR; lanes 3, 1 nM EcFrmR tetramer; lane 4, 1 nM EcFrmR tetramer pre-treated with 200-molar excess formaldehyde. The locations of the frmR and ndh are indicated.
