The main focus of this
project is on the Pyrococcus heat shock regulator, Phr.
It was described to be the first archaeal regulator of heat shock response
(Vierke, G., Engelmann, A., Hebbeln, C. and Thomm, M. (2003) J. Biol.
Chem. 278, 18-26).
The regulator Phr represses the transcription of the heat shock genes
hsp 20 and of aaa+ atpase. Furthermore Phr inhibits the transcription
of its own gene.
EMSAs and footprinting data were used to analyse the mechanism of regulation.
They show that Phr binds to the promoter downstream of the transcription
factors TBP and TFB. Phr overlaps the transcription start site and inhibits
the association of the polymerase and the transcription of the gene.
the crystal structure of Phr was solved (Liu W., Vierke G., Wenke A.,
Thomm M. and Ladenstein R. (2007) J. Mol. Biol. 369, 474-488).
The N-terminal part of Phr consists of a winged helix DNA binding domain
(wH-DBD) and the C-terminal part of an antiparallel coiled coil helical
To obtain more information
about the heat shock response of Pyrococcus furiosus all genes
regulated by Phr should be identified. In vitro transcription
and microarray analysis were conducted functional in the ROMA approach
(Thomm (2007) Archaea: Molecular and Cellular Biology, Edited by Ricardo
Cavicchioli, ASM Press, Washington, D.C.)
Restricted chromosomal DNA of Pyrococcus furiosus was transcribed
in vitro in the presence and absence of Phr. The RNA was labelled
with different fluorescent dyes and hybridized to a whole genome microarray.
The transcription of the heat shock genes for example was repressed
in the presence of the repressor in the transcription reaction. The
comparison of transcriptions with and without regulator allowed the
identification of regulated genes.
The microarray analysis was done in cooperation with Dr. Gerrit Schut,
Laboratory of Prof. Dr. Mike Adams, University of Georgia, Athens.