A Cell-Free Transcription System for the Hyperthermophilic Archaeon Pyrococcus Furiosus

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Abstract
We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vitro initiation of transcription. Pyrococcus transcriptional components were separated into at least two distinct transcription factor activities and RNA polymerase. One of these transcription factors could be functionally replaced by Methanococcus aTFB and Thermococcus TATA bind- ing protein (TBP). Immunochemical analyses demonstrated a structural relationship between Pyrococcus aTFB and Thermococcus TBP. These findings indicate that a TATA box and a TBP are essential components of the Pyrococcus transcriptional machinery.