Regulation of Transcription in Pyrococcus
Isabell DAHLKE*a, Torsten LAMMERSa, John van der OOSTb, Kathleen SANDMANc, Greti SCHUTb, Judith TUININGAb, John REEVEc, Carina HETHKEa, Willem de VOSb, and Michael THOMMa
a Institut fur Allgemeine Mikrobiologie, Kiel, Department of Microbiology, Am Botanischen Garten 1-9, D-24118 Kiel Germany
b Wageningen Agricultural University, Hesselink van Suchtelenweg 4, NL-6307 CT Wageningen, The Netherlands
c Ohio State University, Department of Microbiology, Columbus OH 43210, USA
To study regulation of gene transcription in a hyperthermophile, the transcription of Pyrococcus furiosus celB gene encoding aβ-glucosidase was analyzed using a Pyrococcus cell-free transcription system. This gene was selected as a model template since its expression is controlled on the transcriptional level in vivo and is strongly stimulated by growth on cellobiose as carbon source. The transcription start site of the celB gene was located 25 bp downstream of a typical archaeal TATA-box. The in vitro activity of the celB promoter was very low compared to other bona fide Pyrococcus promoters suggesting that a positive regulator is involved in expression of this gene.
A homologue of the bacterial regulator Leucine responsive protein (Lrp), detected in the Pyrococcus genome, was expressed in Escherichia coli. The recombinant Lrp was found to inhibit transcription from Pyrococcus promoters, suggesting that bacterial-type of regulators can modulate the activity of the eukaryotic-like transcriptional machinery of Pyrococcus. The histone rHPyA1 from Pyrococcus species GB-3a reduced cell-free transcription from the P. furiosus glutamate dehydrogenase promoter in a manner that was dependent on the histone to DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to histone binding than from a linear template consistent with histone binding introducing physical barriers to RNA polymerase and causing changes in the topology of circular templates that also reduce transcription.
