Inhibition of transcription factor IIIA-DNA interactions by xenobiotic metal ions

Transcription factor IIIA (TFIIIA), a cysteine-rich regulatory protein, is the prototype for the largest known superfamily of eukaryotic transcription factors. Members of the TFIIIA superfamily contain Cys(2)His(2) zinc finger domains responsible for nucleic acid binding. Xenobiotic metal ions, which lack known biological function, were previously used as probes for the structure and function of steroid hormone receptors which contain Cys(2)Cys(2) zinc finger domains. Structural alterations in cysteine-rich regulatory proteins by such ions in viva might potentiate carcinogenesis and other disease processes. In the present study cadmium and other xenobiotic metal ions were used to probe the structure and function of TFIIIA. The specific interaction of TFIIIA with the internal control region (ICR) of the 5S RNA gene, as assayed by DNase I protection, was inhibited by Cd2+ ion concentrations of greater than or equal to 0.1 mu M. Aluminum ions were also found to inhibit the TFIIIA-SS RNA gene interaction, albeit at higher concentrations (greater than or equal to 5 mu M). Inhibition by either metal ion was not readily reversible. Other xenobiotic metal ions, such as mercury or cesium, were not found to be inhibitory under these conditions. None of these ions at the concentrations used in this study affected the ability of DNase I to digest DNA or restriction enzymes to specifically cleave DNA. Preincubation of TFIIIA bound to 5S RNA with either Cd2+ or Al3+ resulted in subsequent DNA binding upon dilution and RNA removal, whereas preincubation of free TFIIIA with the metal ions resulted in inhibition of subsequent DNA binding. Because 5S rRNA also binds the TFIIIA zinc finger domains, these results indicate that the 5S RNA bound to TFIIIA protects the protein from metal inhibition and implicates the zinc fingers in the inhibition mechanism. The nature of the footprint inhibition indicates that the N-terminal fingers of TFIIIA are affected by the metal ions, Cd2+ and Al3+ ions also inhibited the ability of TFIIIA to bind complementary single-stranded DNA and promote renaturation, as measured by Tris-phosphate agarose gel electrophoresis. This gel assay is sensitive to DNA conformation and Al3+ ions were found to alter the conformation of single- and double-stranded DNA in this assay. The inhibition of TFIIIA function in vitro by xenobiotic metals offers new insights into the structure and function of TFIIIA and TFIIIA-type zinc finger proteins. Inhibition by Cd2+ occurs at much lower concentrations than previously observed with steroid hormone receptors and suggests that Cys(2)His(2) zinc finger proteins may be especially sensitive to such agents in vivo.