The transcription of the 5S RNA genes of the African frog Xenopus is a leading model for the regulation of transcription of eukaryotic genes. Transcription of 5S RNA genes is activated by the binding of a protein, transcription factor A, to a control region located in the center on the 120 nucleotide gene for the structural RNA. This transcription factor can be purified in high yield on the basis of the fact that it binds 5S RNA in a storage particle that accummulates in immature oocytes. Preliminary experiments suggest that a symmetrical nucleotide sequence is recognized as the site for the cooperative binding of two molecules of the transcription factor to the gene. The experiments proposed here will produce a collection of single point mutants to allow detailed analysis of the sequences in both duplex DNA and single stranded 5S RNA that are required for binding to the transcription factor. Rapid assay methods are proposed to compare the effects of mutations on both the binding of this factor and in vitro transcription. Additional methods will be developed to study binding of RNA to the transcription factor. Since this transcription factor activates 5S RNA genes for transcription and binds to the 5S RNA product, a detailed understanding of the binding stoichiometry and contact sites is essential to determine whether feedback regulation of transcription is a plausible control mechanism. Previous experiments have shown that cloned 5S RNA genes can be asembled in vitro into stable transcription complexes that persist through many rounds of transcription and provide a model for programmed genes in transcriptionally active chromatin. Additional experiments will attempt to further purify components of the in vitro transcription system in order to study the events required to establish and maintain these stable transcription complexes. Radioactive labeling and maintain these stable transcription complexes. Radioactive labeling and immunological detection methods will be employed to determine whether transcription factor A remains bound to the DNA template during the transcription cycle or whether a molecule of the factor leaves the transcription complex in association with the nascent transcript. Similar methods may permit studies of the stability of transcription complexes during replication as well.
