Identification of a transcription factor IIIA-interacting protein

Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcri ption in eukaryotes, The protein from vertebrates has nine contiguous Cys,H is, zinc fingers which function in nucleic acid binding, and a C-terminal r egion involved in transcription activation. In order to identify protein pa rtners for TFIIIA, yeast two-hybrid screens were performed using the C-term inal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a s ource of potential partners. A cDNA clone was identified which produced a p rotein in yeast that interacted with Xenopus TFIIIA but not with yeast TFII IA, This rat clone was sequenced and the primary structure of the human hom olog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined fro m expressed sequence tags. In vitro interaction of recombinant human TFIIIA -intP with recombinant Xenopus TFIIIA was demonstrated by immunoprecipitati on of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIII A with rat TFIIIA-intP was indicated by co-chromatography of the two protei ns on DEAE-5PW following fractionation of a rat liver extract on cation, an ion and gel filtration resins, In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xen opus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.