Human TFIIIA alone is sufficient to prevent nucleosomal repression of a homologous 5S gene

Plasmid DNA harbouring the human 5S rRNA gene was assembled into nucleosomes using either Xenopus S150 extracts or purified core histones in the presence of pectin. In both cases reconstitution of nucleosomes led to a complete repression of transcription. This repression could be efficiently counteracted by preincubating the template DNA with highly purified hTFIIIA which allowed the protein to bind to the ICR of the 5S gene. By using an efficient and well-defined in vitro reconstitution system based on isolated core histones in the presence of pectin, which is devoid of endogenous transcription factors, we demonstrate here for the first time that human TFIIIA alone is sufficient to prevent nucleosomal repression of h5S gene transcription and that additional pol III transcription factors are not required to achieve this effect. Additionally, we investigated the binding of hTFIIIA to a mononucleosome reconstituted on the human 5S gene. DNAse I footprinting experiments reveal that the entire ICR of the human 5S gene is covered by the nucleosome, thereby precluding the subsequent binding of human TFIIIA to the promoter of the 5S gene.