Mutations in 5S DNA and 5S RNA have different effects on the binding of Xenopus transcription factor IIIA

The effects on TFIIIA binding affinity of a series of substitution mutations in the Xenopus laevis oocyte 5S RNA gene were quantified. These data indicate that TFIIIA binds specifically to 5S DNA by forming sequence-specific contacts with three discrete sites located within the classical A and C boxes and the intermediate element of the internal control region. Substitution of the nucleotide sequence at any of the three sites significantly reduces TFIIIA binding affinity, with a 100-fold reduction observed for substitutions in the box C subregion. These results are consistent with a direct interaction of TFIIIA with specific base pairs within the major groove of the DNA. A comparison of the TFIIIA binding data for the same mutations expressed in 5S RNA indicates that the protein does not make any strong sequence-specific contacts with the RNA. Although the protein footprinting sites on the 5S DNA and 5S RNA are coincident, nucleotide substitutions in 5S RNA which moderately reduce TFIIIA binding affinity do not correspond at all to the three specific TFIIIA interaction sites within the gene. The implications of these results for models which attempt to reconcile the DNA and RNA binding activities of TFIIIA by proposing a common structural motif for the two nucleic acids are discussed.     

Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.     

A difference in the importance of bulged nucleotides and their parent base pairs in the binding of transcription factor IIIA to Xenopus 5S RNA and 5S RNA genes

Individual bulge loops present in Xenopus 5S RNA (positions 49A-A50 in helix III, C63 in helix II and A83 in helix IV), were deleted by site directed mutagenesis. The interaction of these mutant 5S RNA molecules with TFIIIA was measured by a direct binding assay and a competition assay. The results of these experiments show that none of the bulged nucleotides in Xenopus 5S RNA are required for the binding of TFIIIA. The affinity of the mutant 5S RNA genes for TFIIIA was also studied by a filter binding assay. In contrast to the effect that deleting bulged nucleotides had on the TFIIIA-RNA binding affinity, deletion of the corresponding A-T base pair at position +83 in 5S DNA was found to reduce the apparent association constant of TFIIIA by a factor of four-fold.