Journal: Nature Communications

Article Title: Tetrameric architecture of an active phenol-bound form of the AAA + transcriptional regulator DmpR

doi: 10.1038/s41467-020-16562-5

Figure Lengend Snippet: a Positions of phenol and zinc inside the core (β/α) 4 barrel scaffold of the sensory domain. b Key residues in the phenol-binding pocket and residues involved in zinc coordination are shown as sticks. c Depiction of the hydroxyl group of phenol interacting with His100 and Trp128 (left); sequence alignment of five aromatic compound-binding transcriptional activators (right). DmpR, P. putida KCTC 1452; PoxR, Ralstonia sp . E2; MopR, Acinetobacter guillouiae ; and XylR, Pseudomonas putida . d The interface of the coiled-coil B-linkers with the strips of leucine residues highlighted within the enlarged box. e B-factor putty tube representation of the B-linker in the ATPase domain connection loop. Orange and red colours and a wider tube indicate regions with high B-factors, whereas shades of green and narrower tubes indicate regions with low B-factors. The putative ATP-binding site is shown in dot representation.

Article Snippet: To determine change of DmpR tetramer by ATP analogue or UAS containing DNA, 3 mM ATP analogue (AMP-PNP/ATPγS), 10 nM DNaseI (NEB) or 20 µM cognate DNA with the UAS sites were co-incubated with DmpR for 20 minutes prior to BN-PAGE analysis.

Techniques: Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Tetrameric architecture of an active phenol-bound form of the AAA + transcriptional regulator DmpR

doi: 10.1038/s41467-020-16562-5

Figure Lengend Snippet: a Schematic diagram of wild type DmpR (DmpR WT ) and truncated DmpR proteins (DmpR ΔD , residues 18–481; DmpR C , residues 232–481; DmpR BC , residues 205–481; and DmpR ΔS , residues 205–563). The molecular weights given include the N-terminal 6 × His tag. b BN-PAGE of truncated DmpR proteins after the addition of ATP or ATPγS. Bands corresponding to tetramers are marked by an arrow. Asterisks indicate an artefact band of DmpR ΔS . This data is representative of three replicates with similar results. c SEC combined with multi-angle light scattering (MALS) analysis to calculate the molecular weight (MW) of DmpR ΔS (dotted black line). d ATPase activity of DmpR WT and DmpR ΔD in the presence or absence of phenol. Hydrolysis of [γ-P ] ATP to generate P was visualised by thin-layer chromatography. ‘C’ represents the reaction with the reaction buffer as control. ‘P’ represents the reaction with the alkaline phosphatase as positive control. This data is representative of five replicates with similar results. e ATPase activities of truncated DmpR derivatives. This data is representative of five replicates with similar results.

Article Snippet: To determine change of DmpR tetramer by ATP analogue or UAS containing DNA, 3 mM ATP analogue (AMP-PNP/ATPγS), 10 nM DNaseI (NEB) or 20 µM cognate DNA with the UAS sites were co-incubated with DmpR for 20 minutes prior to BN-PAGE analysis.

Techniques: Molecular Weight, Activity Assay, Thin Layer Chromatography, Positive Control

Journal: Nature Communications

Article Title: Tetrameric architecture of an active phenol-bound form of the AAA + transcriptional regulator DmpR

doi: 10.1038/s41467-020-16562-5

Figure Lengend Snippet: a Illustration of a single-molecule TIRF assay of eGFP-DmpR binding to σ 54 (1–119) -CPD. b The number of eGFP-DmpR complexes bound to σ 54 (1–119) -CPD without/with phenol and ATPγS per field of view (25 × 50 μm 2 ). Data are presented as mean ± SD from five independent experimental replicates. c A representative time trace of tetrameric eGFP-DmpR binding to surface-immobilized σ 54 (1–119) -CPD. d Distribution of photobleaching steps of the eGFP-DmpR complexes bound to σ 54 (1–119) -CPD. Data represent the mean ± SD from three independent experimental replicates with n ≥ 980 individual molecule (Counts). Events with more than eight photobleaching steps were categorised as aggregates (A). e Schematic representation of a single-molecule TIRF assay of σ 54 (1–119)) -CPD binding to surface-immobilized eGFP-DmpR. f A representative time trace of Cy5-labelled σ 54 (1–119) -CPD binding to antibody-tethered tetrameric eGFP-DmpR. Laser excitation (637 nm followed by 473 nm) is indicated above the trace. The photobleaching time point of the Cy5 signal is indicated. The photobleaching steps of eGFP-DmpR are represented by the dotted line (purple). g Distribution of photobleaching steps of the eGFP-DmpR complexes. eGFP signals co-localized with the signals of Cy5-labelled σ 54 (1–119) -CPD were analysed. Data represent the mean ± SD from three independent experimental replicates with n ≥ 185 individual molecule (Counts). Events with more than eight photobleaching steps were categorised as aggregates (A).

Article Snippet: To determine change of DmpR tetramer by ATP analogue or UAS containing DNA, 3 mM ATP analogue (AMP-PNP/ATPγS), 10 nM DNaseI (NEB) or 20 µM cognate DNA with the UAS sites were co-incubated with DmpR for 20 minutes prior to BN-PAGE analysis.

Techniques: Binding Assay