An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated.
The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
Brinkman
A B
AB
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. arjen.brinkman@algemeen.micr.wau.nl
Dahlke
I
I
Tuininga
J E
JE
Lammers
T
T
Dumay
V
V
de Heus
E
E
Lebbink
J H
JH
Thomm
M
M
de Vos
W M
WM
van Der Oost
J
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D001665:Binding Sites; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D019848:Gene Expression Regulation, Archaeal; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D020027:Pyrococcus furiosus / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; C418107:LrpA protein, Pyrococcus furiosus; D011994:Recombinant Proteins; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein