Cloning, expression, and structural characterization of the n-terminal "zinc ribbon" domain of transcription factor b from various archaea
Colangelo, Christopher Michael
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Transcription factor (II)B (TF(II)B) is conserved throughout evolution from archaea to eucarya and is an essential component in transcription initiation of protein coding genes. It contains multiple functional domains and has been shown to interact with DNA, two general transcription factors and RNA polymerase II. The N-terminal domain contains a Cys-X2-(Cys/His)-X14–17-Cys-X2-Cys motif that was proposed to bind metal. Therefore, using both spectroscopic and biochemical techniques, the N-terminal domain of TF(II)B from both archaeal and eucaryal sources was cloned, expressed and structurally characterized. For example, the 49 N-terminal residues of Pyrococus furiosus TFB (PfTFB) were cloned, expressed, and purified to homogeneity. Then using ICP-AES, XAS, and NMR we probed the metal binding properties of the Cys-X2-Cys-X14-Cys-X2-Cys motif and showed that this protein binds zinc and also folds into a "zinc ribbon" domain. Subsequent analysis on the N-terminal domain of both human TFIIB and Haloferax volcanii TFB2, showed that these proteins also bind zinc in a similar fashion toPfTFB and most likely possess a similar "zinc ribbon" domain structure. Finally, some initial work on the N-terminal domain of Haloferax volcanii TFB3 proved that this domain is incapable of binding metal, suggesting that it functions differently than the "zinc ribbon" type TF(II)Bs.