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. 2010 Oct 21;39(4):1294–1309. doi: 10.1093/nar/gkq986

Figure 7.

Figure 7.

Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under PAra promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.