Restoration of binding of oxidized transcription factor IIIA to 5S RNA by thioredoxin.

7S particles from Xenopus oocytes were completely dissociated under non-reducing conditions. Studies using glycerol gradient centrifugation show that unlike the native 7S particle in which 5S RNA and TFIIIA co-sedimented in a fairly sharp peak, the RNA from the denatured 7S sedimented at the position corresponding to the 5S RNA and the TFIIIA sedimented as a wide peak between 6S and 12S. Thioredoxin from E. coli can catalyze the reactivation of the TFIIIA as measured by its ability to reform the 7S particle. The rate of reactivation with thioredoxin was significantly greater than with dithiothreitol. Oxidized thioredoxin was unable to reactivate TFIIIA. Pure TFIIIA can be inactivated and subsequently reactivated in the same way by formation of a cross-linked structure via intermolecular disulfide bridges.     

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.