RELATIVE CONTRIBUTIONS OF THE ZINC FINGERS OF TRANSCRIPTION FACTOR IIIA TO THE ENERGETICS OF DNA-BINDING

We have expressed and purified a series of recombinant zinc finger pol ypeptides derived from the cDNA for the Xenopus 5 S gene-specific tran scription factor TFIIIA. Dissociation constants for the interaction of each of the truncated polypeptides with the 5 S gene promoter have be en measured using gel mobility shift assays. DNase I footprinting and proteolysis experiments provide additional insights into the interacti ons of individual fingers within complexes of the truncated proteins. These results are discussed in terms of recently proposed models for t he TFIIIA-DNA interaction. The effects of mutations in two of the stro ngly binding proteins, zfl-3 and zfl-7, on DNA binding affinity have b een investigated. Mutations have been made both in putative DNA-contac t residues and in the linker regions between zinc fingers. The observe d decreases in binding affinity cannot be explained simply in terms of loss of protein-DNA contacts. Our results support a model in which DN A binding is accomplished through sets of interacting zinc fingers tha t make different energetic contributions to the overall binding of the protein and different contacts with the DNA.