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J. Biol. Chem., Vol. 277, Issue 36, 32659-32667, September 6, 2002
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From the Institute of Genetics, University of Bayreuth,
Universitaetsstrasse 30, Bayreuth D-95440, Germany
The hrcA gene of Bacillus
subtilis codes for a transcriptional repressor protein that
negatively regulates expression of the heptacistronic dnaK
and the bicistronic groE operon by binding to an
operator-element called CIRCE. Recently, we have published data
suggesting that the activity of HrcA is modulated by the GroE
chaperonin system. Biochemical analyses of the HrcA protein have been
hampered so far by its strong tendency to aggregate. Here, a genetic
method was used to isolate mutant forms of HrcA with increased activity
under conditions of decreased GroE function. One of these mutant forms
(HrcA114) containing five amino acid replacements exhibited enhanced
solubility when overexpressed. HrcA114 purified under native conditions
produced two retarded CIRCE-containing DNA fragments in band shift
experiments. The amount of the larger fragment increased after addition
of GroEL, GroES, and ATP but decreased when ATP was replaced by the
nonhydrolyzable ATP analog ATP
Isolation and Analysis of Mutant Alleles of the Bacillus
subtilis HrcA Repressor with Reduced Dependency on GroE
Function*
, and
S. DNase I footprinting experiments
exhibited full protection of the CIRCE element and neighboring
nucleotides in an asymmetric way. An in vitro binding assay
using affinity chromatography showed direct and specific interaction
between HrcA114 and GroEL. All these experimental data are in full
agreement with our previously published model that HrcA needs the GroE
chaperonin system for activation.
*
This work was supported in part by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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. Tel.: 49-921-554305;
Fax: 49-921-552710; E-mail: thomas.wiegert@uni-bayreuth.de.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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