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[Preprint]. 2024 Jan 22:2023.01.30.526351. Originally published 2023 Jan 31. [Version 4] doi: 10.1101/2023.01.30.526351

FIGURE 2.

FIGURE 2

DNA binding by PlzA is c-di-GMP dependent. (a) EMSA with increasing concentrations of PlzAWT showing binding to glpFKD(−219) DNA. The mixture was incubated for 10 min before electrophoresis. (b) The same EMSA mixture as in (a) but with a final concentration of 2.5 ng/μL of the nonspecific competitor poly-dI-dC. Protein was incubated with poly-dI-dC for 5 min prior to the addition of labeled glpFKD(−219) probe. (c) The same EMSA mixture as in (b) but supplemented with 100 μM c-di-GMP. Protein was incubated with c-di-GMP for 5 min prior to the addition of any nucleic acids. Arrows indicate PlzA- glpFKD(−219) complexes. Lane 1 in (a-c) is the probe only control, containing 50 nM of probe. Protein concentrations are as follows in each subsequent lane: Lane 2: 0.5 μM, Lane 3: 1 μM, Lane 4: 2.5 μM, Lane 5: 5 μM, Lane 6: 7.5 μM, and Lane 7: 10 μM. (d), (e), (f) EMSA experiments as in (a), (b), and (c), respectively, but performed with the mutant PlzARD-RD protein. All conditions were performed identically as with PlzAWT. Each panel is a representative gel from separate EMSAs which were performed in triplicate (PlzAWT) or duplicate (PlzARD-RD).