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Mol Microbiol. 2000 Dec;38(5):1093-103.

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressordagger.

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  • 1Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester, M60 1QD, UK.

Abstract

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel-shift assays. This requirement is independent of the co-chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein-DNA complex can be 'supershifted' by anti-DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK-HspR-DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole-cell extracts, DnaK was shown to co-purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR-mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co-repressor by binding to HspR at its operator sites. In this model, the DnaK-HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK-sigma32 and Hsp70-HSF systems of other organisms).

PMID:
11123682
[PubMed - indexed for MEDLINE]
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