| In vivo and In vitro Studies of Class I Heat Shock Protein Regulation in Bacillus subtilis |
| Jaclyn Winter, Scott Leddon, Jason Randall, Jason Plyler, Steve Persaud, and Matthew Fountain |
| Class I heat shock proteins in prokaryotic
organisms are negatively regulated by the protein HrcA. HrcA binds to a DNA
inverted repeat, CIRCE, in the promoter region of the GroE operon gene, which
inhibits the transcription of the chaperone proteins. The GroE chaperonin system
modulates the activity of HrcA. Under heat shock conditions, the chaperonin
system is overwhelmed with denatured proteins and is unable to maintain HrcA in
its active form. HrcA dissociates from CIRCE allowing for transcription of the
GroE operon. We characterized the CIRCE inverted repeat sequence to determine
whether or not it adopts a cruciform structure using nuclease S1 digestion,
fluorescence resonance energy transfer, and optical melting studies. These
studies show the CIRCE sequence adopts a linear rather than a cruciform structure
as previously hypothesized. The HrcA gene from B. subtilis was isolated using
PCR, and incorporated into a P-Lex expression vector. A reporter plasmid
containing the GroE promoter, CIRCE sequence and a b-galactosidase reporter gene
was also constructed. The plasmids were transformed into E.coli, which lacks
the HrcA/CIRCE regulatory system. The regulation of the b-galactosidase gene
was studied under different heat shock conditions. This system allows us to
study the thermostatic control of the HrcA:CIRCE complex in vivo and permits us
to expand our studies to include CIRCE sequences and HrcA proteins from other
organisms. |
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