Nucleic Acids Research, Vol 24, Issue 5 924-930, Copyright © 1996 by Oxford University Press
JS Hanas and CG Gunn
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 Cys2His2 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 Cys2Cys2
zinc finger domains. Structural alterations in cysteine-rich regulatory
proteins by such ions in vivo 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 > or = 0.1 microM. Aluminum ions were also found to
inhibit the TFIIIA-5S RNA gene interaction, albeit at higher concentrations
(> or = 5 microM). 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 Cys2His2 zinc finger proteins may be
especially sensitive to such agents in vivo.
ARTICLES
Inhibition of transcription factor IIIA-DNA interactions by xenobiotic metal ions
Department of Biochemistry and Molecular Biology, University of Oklahoma College of Medicine, Oklahoma City 73190, USA.
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