Zn- Cd- and Pb-transcription factor IIIA: properties DNA binding and comparison with TFIIIA-finger 3 metal complexes

摘要

Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn²⁺ from the protein. Zn–TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn²⁺ was shown to depend on the details of the isolation process. The average dissociation constant of Zn²⁺ in Zn-TFIIIIA was 10⁻⁷. The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 × 10⁻⁸. The reactivity of Zn–TFIIIA with a series of metal binding ligands, including 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N⁴-dimethylthiosemicarbazone) (H2KTSM2) revealed similar kinetics. The reactivity of PAR with Zn–TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA. Both Cd²⁺ and Pb²⁺ disrupt TFIIIA binding to its cognate DNA sequence. The Pb²⁺ dissociation constant of Pb-F3 was measured as 2.5 × 10⁻⁸. According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb²⁺.