Effects of zinc finger mutations on the nucleic acid binding activities of Xenopus transcription factor IIIA

Abstract

Transcription factor IIIA (TFIIIA) is required for the activation of 5S RNA gene transcription as well as the storage of 5s RNA as a 7S ribonucleoprotein particle. Interaction with both nucleic acids is mediated through nine C2H2 zinc fingers. In order to determine amino acid regions necessary for nucleic acid interaction, a series of substitution mutants Xenopus laevis TFIIIA have been constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay. All of the mutant TFIIIA proteins retained full 5S RNA binding activity. Substitution of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein with 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. The results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the alpha-helical regions of finger 2 and particularly of finger 3 provide the majority of the free energy of the TFIIIA-DNA interaction. Basic amino acids found at the same position within the alpha-helices of fingers 2 and 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 protein-nucleic acid interactions.