Fig. 8: Functional characterization of CvkR DNA-binding residues.
a Upper left panel: Superimposition of CvkR onto the HiNmlR-promoter complex. Two subunits of the CvkR homodimer are colored in blue or cyan. HiNmlR homodimer and its target DNA in the HiNmlR-promoter complex (PDB code 5D8C) are displayed in gray and orange, respectively. Three regions directly participating in promoter DNA binding in the HiNmlR-promoter complex, helix α2 and two winged loops W1 and W2, are labeled. Lower left panel: electrostatic surface representation of CvkR highlighting the potential electropositive (blue) DNA-binding surface. Right panel: The key DNA-interacting residues reported in HiNmlR are shown in sticks and labeled in gray. The possible DNA-interacting residues in CvkR corresponding to the three DNA-interacting regions are labeled in black. b EMSA assays showing the binding of CvkR variants to Pcas12k. The 33-nt Pwt probe was used for the assay. The His-SUMO tags were removed from all CvkR variants used in the EMSA assays. The free probe and Pwt-protein complexes are marked as “Free” and “Bound”, respectively. The data are representative from three independent experiments. c CvkR and mutant proteins were expressed in the TXTL system37 from pET28a(+) or pET28a(+)-SUMO and detected by Western blot via their N-terminal 6xHis tag. The stained membrane is shown below. The corresponding molecular masses are 29.9 kDa for 6xHis-SUMO-CvkR WT2 and R42E, while 6xHis-TEV-CvkR WT and 6xHis-TEV-CvkRmut have masses of 19.9 and 19.5 kDa. d TXTL assay to test the regulatory capacity of CvkRmut and R42E compared to CvkR (WT and WT2 as defined in panel C) for repressing deGFP fluorescence expressed from the cas12k promoter (Pcas12k). The Pcas12k-deGFP cassette was expressed from plasmid p70a (5 nM) in the absence or presence of 1 pM pET28a(+) expressing CvkRmut, R42E, CvkR WT or WT2. The TXTL reactions were performed at 29 °C overnight, and fluorescence was measured every 10 min in a Wallac 1420 Victor2 microplate reader. Error bars show standard deviations derived from two technical replicates in one representative example of three independent experiments. Source data are provided as a Source Data file.
