Nucleic Acids Research, Vol 24, Issue 13 2567-2574, Copyright © 1996 by Oxford University Press
SJ McBryant, B Gedulin, KR Clemens, PE Wright and JM Gottesfeld
Zinc finger proteins of the Cys2His2 class are DNA sequence-specific
transcription factors. Previous structural studies of zinc finger
protein-DNA complexes have shown that amino acids in the finger tip and
alpha-helix regions within individual finger domains make base-specific
contacts with the major groove of DNA. The nine finger protein
transcription factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base
pair region of the 5S RNA gene through major groove interactions with two
sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous
studies have suggested that zinc fingers 4 and 6 each bind in or across the
minor groove to bridge these major groove-binding zinc fingers. Here it is
shown that a polypeptide containing zinc fingers 1- 5 (zf1-5) binds
oligonucleotides with modifications in the major groove of the finger 4
binding site with wild-type affinity. Mutagenesis and binding site
selection studies were performed to determine whether high affinity DNA
binding by zf1-5 requires a particular sequence in the binding site for
finger 4. Several mutations in this region of the 5S gene reduced the
DNA-binding affinity of zf1-5; however, selection and amplification binding
assays did not recover the wild-type finger 4 binding site sequence from a
pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on
the top strand was highly selected within the finger 4 binding site. We
suggest that high affinity DNA binding by zinc finger 4 may be dictated by
a sequence-specific DNA structure rather than by a unique DNA sequence.
Deletion of finger 4 from zf1-5 results in a protein with poor binding
affinity, demonstrating the importance of finger 4 in proper alignment of
neighboring fingers with the DNA, and/or the importance of correct
protein-protein interactions between fingers.
ARTICLES
Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
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