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Molecular mechanism of transcriptional regulation of the heat shock response in archaea
All living organisms share a common molecular stress response upon rapidly up-shifted environmental temperature. The heat shock response is characterized by a dramatic change in gene expression patterns and elevated syntheses of a family of heat shock proteins, most of which function as molecular chaperones in preventing the aggregation of denatured proteins and/or helping protein refolding. The expression of most heat shock genes is strictly repressed under normal conditions, but activated once stress response is triggered. The mechanism of heat shock regulation differs among the three kingdoms. Compared to bacteria and eukaryotes little is known about heat shock regulation in archaea.
The first transcriptional regulator selectively inhibiting cell-free transcription of archaeal heat shock promoters has been recently identified from the hyperthermophilic archaeon P. furiosus (1). The 24 kDa protein named as Phr forms a homodimer and specifically inhibits transcription in vitro and in vivo by binding to a 29-bp DNA sequence overlapping the transcription start site in heat shock promoters. Recently the crystal structure of Phr was solved by the MAD technique at 2.6 Å. The refined model revealed a stable homodimer, each subunit consisting of a N-terminal winged helix DNA-binding domain (wH-DBD) and a C-terminal antiparallel coiled coil helical domain. The overall structure shows as a molecular chimera with significant folding similarity of its DBD to the bacterial SmtB/ArsR family, while its C-terminal part was found to be a remote homologue of the eukaryotic BAG domain. Molecular docking and mutational analyses suggested a novel DNA binding mode in which the major specific contacts occur at the minor groove. Nonetheless, we have little knowledge on how the cell works to derepress the modulated genes under stress in Pyrococcus.
The major aims of our studies
1. Screen for proteins in P. furiosus that possibly interact with the C-terminal domain of Phr, and characterize these proteins.
2. Crystallize the protein-DNA complex using the strategy of covalent trapping by introducing an artificial disulfide bond between the Phr protein and the oligonucleotide.
Possible job for students
• Site-directed mutagenesis on Phr and purification of the mutants.
• Mass spectrometry analyses of the possible Phr binding proteins, cloning of the genes encoding these proteins and probably expression and purification of them.
1. Vierke, G., Engelmann, A., Hebbeln, C. & Thomm, M. (2003). A novel archaeal transcriptional regulator of heat shock response. J Biol Chem 278, 18-26.
2. Liu, W., Vierke, G., Wenke, AK. Thomm, M. & Ladenstein R. (2007). Crystal structure of the archaeal heat shock regulator from Pyrococcus furiosus. A molecular chimera representing eukaryal and bacterial features. J. Mol. Biol. (in press)
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