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(Received for publication, August 12, 1996, and in revised form, October 2, 1996)
From the Department of Molecular Biology and Biochemistry,
University of California, Irvine, California 92697
Binding of cAMP receptor protein (CRP) and CytR
mediates both positive and negative control of transcription from
Escherichia coli deoP2. Transcription is activated by CRP
and repressed by a multi-protein CRP·CytR·CRP complex. The latter
is stabilized by cooperative interactions between CRP and CytR. Similar
interactions at the other transcriptional units of the CytR regulon
coordinate expression of the transport proteins and enzymes required
for nucleoside catabolism. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this
sort regulate differential expression. To understand the combinatorial
control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states. By comparison to distributions for
other CytR-regulated promoters, we hope to understand the roles of
individual states in differential gene expression. The results indicate
that CytR binds specifically to multiple sites at deoP2,
including both the well recognized CytR site flanked by CRP1 and CRP2
and also sites coincident with CRP1 and CRP2. Binding to these multiple
sites yields both cooperative and competitive interactions between CytR
and CRP. Based on these findings we propose that CytR functions as a
differential modulator of CRP1 versus CRP2-mediated
activation. Additional high affinity specific sites are located at
deoP1 and near the middle of the 600-base pair sequence
separating P1 and P2. Evaluation of the DNA sequence requirement for
specific CytR binding suggests that a limited array of contiguous and
overlapping CytR sites exists at deoP2. Similar extended
arrays, but with different arrangements of overlapping CytR and CRP
sites, are found at the other CytR-regulated promoters. We propose that
competition and cooperativity in CytR and CRP binding are important to
differential regulation of these promoters.
Volume 271, Number 52,
Issue of December 27, 1996
pp. 33242-33255
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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