| The glucose-6-phosphate-inducible Uhp system of Escherichia coli is a member of a broad family of "two-component" signal transduction systems in bacteria responsible for coordinating cellular physiology with alterations in the environment. Expression of the transporter for this system, UhpT, is regulated at the level of transcription by the regulon-specific activator UhpA and the global response regulator CAP. To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter, purified UhpA was phosphorylated with acetyl phosphate. This reaction was dependent upon Mg;Purified CAP was shown to bind to only one site in the uhpT promoter, centered at ;To analyze the mechanism of synergistic action of the two transcription activators, mobility shift experiments and DNase I and hydroxyl radical footprinting experiments were performed. Despite the ability of CAP to produce a conformational change in the uhpT promoter, the concentration-dependent binding of UhpA was not affected by CAP, nor did UhpA alter the DNA-binding properties of CAP. Each transcription activator, however, was able to stabilize RNAP binding to the uhpT promoter. A model of transcription activation at the uhpT promoter is proposed. |
