title: The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressordagger.
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).
authors: Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester, M60 1QD, UK.
authors: G
authors: G
authors: Bucca
authors: A M
authors: AM
authors: Brassington
authors: H J
authors: HJ
authors: Schönfeld
authors: C P
authors: CP
authors: Smith
mesh_terms: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000302:isolation & purification; D018832:Molecular Chaperones / Q000302:isolation & purification / Q000502:physiology*; D011401:Promoter Regions, Genetic; D018085:Regulon; D012097:Repressor Proteins / Q000302:isolation & purification / Q000502:physiology*; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C108444:HspR protein, bacteria; D018832:Molecular Chaperones; D012097:Repressor Proteins; C063842:dnaK protein, E coli