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(Received for publication, July 25, 1996, and in revised form, October 29, 1996)
From the Department of Microbiology, University of Virginia School
of Medicine, Charlottesville, Virginia 22908
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
Volume 272, Number 3,
Issue of January 17, 1997
pp. 1910-1919
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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.
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