A transcription factor IIB homolog from the hyperthermophilic archaeon Pyrococcus furiosus binds Zn or Fe in an N-terminal Cys₄ motif

Abstract

 The gene for an archaeal homolog of the eukaryotic transcription factor TFIIB has been cloned from the marine hyperthermophilic archaeon Pyrococcus furiosus and overexpressed in Escherichia coli. This TFB gene displays a sequence that is identical to a gene sequence in P. woesei. A gene for the 49-residue N-terminal domain of TFB that contains a putative C-X₂-C-X₁₅-C-X₂-C metal-binding motif was subcloned and overexpressed as TFB-NTD. Purification of the TFB-NTD gene product yields Zn- and Fe-containing forms, which have been characterized by mass spectrometry and UV-visible, electron paramagnetic resonance, and X-ray absorption (XAS) spectroscopies. Only the Zn form of the TFB holoprotein has been (partially) purified, and it has been characterized by XAS. All spectroscopic characteristics are consistent with a nearly tetrahedral MS₄ metal-binding site made up of the four cysteine residues in the N-terminal domain. The relatively greater thermal stability of the Zn form suggests that TFB may be a Zn-containing protein involved in archaeal transcription.