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Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in  Pyrococcus furiosus . The protein is located in an operon structure together with the hypothetical gene  pf1089  and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of  tfb-RF1  using a  Pyrococcus  shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the  pf1011/1012  operon, beside the expected target of the  pf1089/tfb-RF1  region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and  in vitro  analysis using gel shift assays, DNase I footprinting and  in vitro  transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with  in vivo  data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of  tfb-RF1  enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in  P. furiosus .

The Transcriptional Regulator TFB-RF1 Activates Transcription of a Putative ABC Transporter in Pyrococcus furiosus