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

Abstract

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 × 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 × 10 −8 . According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb 2+ .

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

“…Methods for preparation of recombinant X. laevis TFIIIA from Escherichia coli and for the isolation of MT-2 from rabbit liver have been described in detail elsewhere [35,36]. Zn 7 -or Cd 7 -MT-2 (Ac-MDPNCSCAADGSCTCATSCKCKECKCTSCKKSCCSCCPSGCAKCAQGCICKGASDK CSCCA) have molecular masses between 6 and 7 kDa and migrate on Sephadex G-75 like a globular 10-kDa protein [37].…”

“…The purity of the TFIIIA protein was shown to be routinely 80-90% according to densitometric analysis of silver-stained 12% SDS-PAGE of various protein preparations [35]. The metal-binding stoichiometry of TFIIIA was found to vary between less than 1-9 for both Zn-and Cd-forms, depending on the details of the preparation procedure and buffering conditions [35].…”

“…The metal-binding stoichiometry of TFIIIA was found to vary between less than 1-9 for both Zn-and Cd-forms, depending on the details of the preparation procedure and buffering conditions [35]. Zn n -TFIIIA (n = 7 ± 2) is active in DNA-binding in buffers containing 5-20 μM of excess Zn 2+ [35]. Since TFIIIA with less than 9 bound metals may be relevant in vivo, protein samples of various metal stoichiometries were used in the present study and the results compared.…”

“…The electrophoretic mobility shift assay (EMSA) as well as the method for the preparation and radio-labeling of the 146 bp ICR-containing DNA probe have been described elsewhere [35]. After electrophoresis for 2 h, the gel was dried and placed in contact with X-ray film (Fuji) at −80 °C to reveal the location and concentration of free and bound ICR.…”

“…Cd 2+ reacts with Zn-TFIIIA-ICR and causes the dissociation of the protein-DNA adduct [35]. Thus, it was of interest to determine whether apo-or Zn-MT might compete successfully for Cd 2+ bound to TFIIIA and, thereby, provide the possibility to restore its DNA-binding capacity (reaction (4)): (5) Fig.…”