Nucleocytoplasmic transport of 5S ribosomal RNA

Nucleocytoplasmic transport of 5S ribosomal RNA in Xenopus oocytes occurs in the context of small, non-ribosomal RNPs. The complex with the zinc finger protein TFIIIA (7S RNP) is exported from the nucleus and stored in the cytoplasm, whereas the complex with the ribosomal protein L5 (5S RNP) shuttles between the nucleus and the cytoplasm. Nuclear import- and export-signals appear to reside within the protein moiety of these RNPs. Import of TFIIIA is inhibited by RNA binding, whereas nuclear transfer of L5 is not influenced by RNA binding. We propose that the export capacity of both, TFIIIA and L5, is regulated by the interaction with 5S ribosomal RNA.     

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.