Structural evidence for a common zinc binding domain in archaeal and eukaryal transcription factor IIB proteins

X-ray absorption spectroscopy has been used to compare the metal coordinati on of the N-terminal zinc binding domain of eukaryal human transcription fa ctor (TF) IIB to the previously reported structure of archaeal Pyrococcus f uriosus (Pf) TFB. Full length and N-terminal fragments for both PfTFB and h uman TFIIB were cloned, expressed, and purified. The [C10H] variant of PfTF B was constructed to resemble the metal binding motif of higher eukaryal TF IIB proteins by mutating the second cysteine ligand to a histidine. All fiv e proteins bind zinc in a 1:1 ratio. Zn X-ray absorption spectroscopy of hu man TFIIB and [C10H]PfTFB mutant are consistent with ZnS3(N,O) ligation, an d further suggest that the N/O ligand is an imidazole.