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 t ranscription factors. Previous structural studies of zinc finger prote in-DNA complexes have shown that amino acids in the finger tip and alp ha-helix regions within individual finger domains make base-specific c ontacts with the major groove of DNA. The nine finger protein transcri ption factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair r egion of the 5S RNA gene through major groove interactions with two se ts 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 acros s 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 bindin g site selection studies were performed to determine whether high affi nity DNA binding by zf1-5 requires a particular sequence in the bindin g site for finger 4, Several mutations in this region of the 5S gene r educed the DNA-binding affinity of zf1-5; however, selection and ampli fication 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 t he finger 4 binding site, We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure ra ther than by a unique DNA sequence. Deletion of finger 4 from zf1-5 re sults in a protein with poor binding affinity, demonstrating the impor tance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions bet ween fingers.