Nucleic Acids Research, 1991, Vol. 19, No. 24 6871-6876
© 1991
MOLECULAR BIOLOGY |
Structural elements in the N-terminal half of transcription factor IIIA required for binding to the 5S RNA gene internal control region
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center Oklahoma City, OK 73190, USA
*To whom correspondence should be addressed
Received August 28, 1991. Accepted November 11, 1991.
Zinc binding domains and the conserved Thr-Gly-Glu-Lys (TGEK) tetrapeptide in the N-terminal half of transcription factor IIIA (TFIIIA) were subjected to in vitro mutagenesis to biochemically assess their role in factor interaction with the 5S gene internal control region (ICR). TFIIIA containing a Leu in place of Hls33 in the Cys2-His2 zinc binding site of finger I lost the ability to protect the entire 5S RNA gene ICR (nucleotides +96 to +43) from DNase I digestion. Thus, mutation of one potential zinc ligand in the Nterminal finger inhibited specific DNA binding by the N-terminal as well as downstream fingers. Cooperativity apparently exists among TFIIIA zinc fingers in metal binding/finger folding and DNA binding. Substituting a Ser for Gly69 or a Glu for Lys 71 in the conserved TGEK tetrapeptide in finger II of TFIIIA resulted in the loss of DNA binding. A Gly-dependent bend structure and a terminal positive charge in this tetrapeptide are important for TFIIIA interaction with DNA. Whereas TFIIIA with a Ser substituted for Cys20 in finger I (proposed zinc ligand) did not protect the ICR from DNase I digestion, TFIIIA containing a Ser substituted for Cys35 (not a proposed zinc ligand) retained the ability to bind the ICR. When Cys112 or Cys 164 (proposed zinc ligands in fingers IV and VI) were replaced by Ser, the DNase I footprint patterns afforded by the espective mutant proteins were similar, protection on the ICR from about nucleotides +96 up to +78. A similar pattern was obtained with a TFIIIA mutant in which fingers V,VI,VII, and a portion of VIM were deleted. Maintenance of zinc coordination spheres is necessary for DNA binding by downstream fingers. The six fingers comprising the N-terminal half of TFIIIA appear to act in two groups of three with binding of the second group dependent upon initial binding of the N-terminal group to the +90 to +80 region of the 5S gene ICR.
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