Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA.
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.
McBryant
S J
SJ
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Gedulin
B
B
Clemens
K R
KR
Wright
P E
PE
Gottesfeld
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009865:Oocytes; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017186:Titrimetry; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D004247:DNA