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

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 Zn(2+) from the protein. Zn-TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn(2+) was shown to depend on the details of the isolation process. The average dissociation constant of Zn(2+) in Zn-TFIIIIA was 10(-7). The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 x 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-(N(4)-dimethylthiosemicarbazone) (H(2)KTSM(2)) 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 Cd(2+) and Pb(2+) disrupt TFIIIA binding to its cognate DNA sequence. The Pb(2+) dissociation constant of Pb-F3 was measured as 2.5 x 10(-8). According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb(2+).