Group Prof. Dr. M. Thomm
Coworkers: Annette Keese


Regulation of heat shock response in Pyrococcus furiosus

 

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.

 

 

Recently 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 domain.

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.