An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated

The archaeal transcriptional initiation machinery closely resembles core el ements of the eukaryal polymerase II system. However, apart from the establ ished basal archaeal transcription system, little is known about the modula tion of gene expression in archaea, At present, no obvious eukaryal-like tr anscriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that po tentially encodes a bacterial-like transcriptional regulator, In the presen t study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation, For that purpose, we ha ve produced LrpA in Escherichia coli, In a cell-free P. furiosus transcript ion system we used wild-type and mutated lrpA promoter fragments to demonst rate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, i n which LrpA appeared to be present in (at least) a tetrameric conformation . The location of the LrpA binding site was further identified by DNaseI an d hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pa ir sequence that overlaps the transcriptional start site of its own promote r. The molecular basis of the transcription inhibition by LrpA is discussed .