Separate contributions of UhpA and CAP to activation of transcription of the uhpT promoter of Escherichia coli

Activation of promoters by multiple transcription factors might occur throu gh favorable contacts of the activators with themselves or RNA polymerase, or by changes in DNA geometry that enhance formation of the transcription c omplex. Transcription of the Escherichia coil uhpT gene, encoding the organ ophosphate transporter, requires the response regulator UhpA and is stimula ted by the global regulator protein CAP. CAP binds to the uhpT promoter at a single site, centered at -103.5 bp relative to the start of transcription and UhpA binds to multiple sites between positions -80 and -32. Overexpres sion of UhpA did not reduce the degree of CAP stimulation of uhpT-lacZ expr ession, showing that CAP action is more complex than enhancement of the bin ding of UhpA. Footprinting experiments demonstrated that UhpA and CAP modes tly stimulated each other's binding to the uhpT promoter, but did not affec t the positioning of the binding sites. An in vitro transcription system wa s used to examine the contribution of each transcription factor at the uhpT promoter. Action of UhpA and CAP proteins was not affected by template sup ercoiling. Kinetic analyses of productive and abortive initiation showed th at CAP acted both to stabilize by fivefold the open promoter complexes form ed in the presence of UhpA and to enhance by twofold the rate of their form ation. These results indicate that open complex formation requires UhpA and that CAP stabilizes the open complex. (C) 1999 Academic Press.