Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter

John L. Dahl, Bei Yang Wei, Robert J. Kadner

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

Expression of the Escherichia coli sugar phosphate transporter UhpT is induced by extracellular glucose 6-phosphate through a transmembrane signaling process dependent on the sensor kinase UhpB and the UhpT homolog, UhpC. These proteins are thought to regulate the phosphorylation of the transcription activator, UhpA. To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter region, the UhpA protein was overexpressed and purified. Purified UhpA was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and on the presence of aspartate 54, the site of phosphorylation in homologous response regulators. Gel electrophoretic mobility shift and DNase I and hydroxyl radical protection assays showed that UhpA bound specifically to the region of the uhpT promoter extending from -80 to -50 bp, relative to the transcription start site. At higher concentrations of UhpA, binding was extended to the -32 region. Binding to the -64 element exhibited positive cooperativity and was stimulated severalfold by phosphorylation of UhpA, whereas extension to the downstream region was more strongly affected by phosphorylation. The consensus sequences for the high affinity UhpA-binding sites in the -64 element and for the downstream, low affinity sites are proposed. The pattern of in vitro binding by UhpA agreed with the in vivo observations that phosphorylation-independent assembly of the transcription initiation complex can occur at elevated concentrations of UhpA.

Original languageEnglish (US)
Pages (from-to)1910-1919
Number of pages10
JournalJournal of Biological Chemistry
Volume272
Issue number3
DOIs
StatePublished - 1997

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