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J. Biol. Chem., Vol. 277, Issue 18, 15773-15780, May 3, 2002
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From the Department of Microbiology, Cornell University,
Ithaca, New York 14853
OccR is a LysR-type transcriptional regulator of
Agrobacterium tumefaciens that positively regulates the
octopine catabolism operon of the Ti plasmid. Positive control of the
occ genes occurs in response to octopine, a metabolite
released from plant tumors. Octopine causes DNA-bound OccR to undergo a
conformational change from an inactive to an active state; this change
is marked by a decrease in footprint length from 55 to 45 nucleotides
as well as a relaxation of a high angle DNA bend. In this study, we
first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to
lock OccR-DNA complexes into a conformation resembling the inactive
conformation, whereas other mutations were designed to lock complexes
into the active conformation. These mutations altered the conformation
of OccR-DNA complexes and their responses to octopine in ways that we
had predicted. As expected, operator mutations that locked complexes
into a conformation having a long footprint and a high angle DNA bend
blocked activation by octopine in vivo. Surprisingly,
however, mutations that lock OccR into a short footprint and low angle
DNA bend failed to cause the protein to function constitutively.
Furthermore, some of the latter mutations interfered with activation by
octopine. We conclude that locking OccR into a conformation having a
short footprint is not sufficient to cause constitutive activation, and
octopine must cause at least one additional conformational change in
the protein.
Mutations in the occQ Operator That Decrease
OccR-induced DNA Bending Do Not Cause Constitutive Promoter
Activity*
*
This work was supported by National Research Service Award
GM41892 from the NIGMS of the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
360A Wing Hall, Cornell University, Ithaca, NY. Tel.: 607-255-2413; Fax: 607-255-3904; E-mail: scw@cornell.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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