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

Frontiers in Microbiology
Robert ReicheltWinfried Hausner

Abstract

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 vitr...Continue Reading

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Datasets Mentioned

BETA
E-MTAB-6618

Methods Mentioned

BETA
immunoprecipitation
PCR
ChIP
electrophoresis
electrophoretic
footprinting
in
Assay
ChIP-seq
in vitro transcription

Software Mentioned

Primer3
Pique
WebLogo
Bowtie2
Rotorgene
Integrative Genome Viewer ( IGV )
IGV genome browser
AIDA Image Analyzer
TMHMM
MEME Suite

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