EFFECTS OF ZINC-FINGER MUTATIONS ON THE NUCLEIC-ACID BINDING ACTIVITIES OF XENOPUS TRANSCRIPTION FACTOR IIIA

Transcription factor IIIA (TFIIIA) is required for the activation of 5 S RNA gene transcription as well as the storage of 5S RNA as a 7S ribo nucleoprotein particle. Interaction with both nucleic acids is mediate d through nine C2H2 zinc fingers. In order to determine amino acid reg ions necessary for nucleic acid interaction, a series of substitution mutants of Xenopus laevis TFIIIA have been constructed and expressed a s recombinant proteins in Escherichia coli. The mutant proteins were p urified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay. All of the mu tant TFIIIA proteins retained full 5S RNA binding activity. Substituti on of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein wi th the 5S RNA gene. In contrast, substitution of finger 1 or finger 7 had little effect on the interaction of TFIIIA with the 5S RNA gene. T he results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the cr-hel ical regions of finger 2 and particularly of finger 3 provide the majo rity of the free energy of the TFIIIA-DNA interaction. Basic amino aci ds found at the same position within the alpha-helices of fingers 2 an d 3 of TFIIIA are required for high-affinity DNA binding activity. The identification of amino acid residues critical for the formation of a TFIIIA-DNA complex contributes to our understanding of zinc finger pr otein-nucleic acid interactions.