9604724 Pape The primary determinant in recognizing and activating the large rRNA gene promoter in eukaryotes is an essential transcription factor for RNA polymerase I (RNAP I), which maintains a species-specific and polymerase-specific complex with the promoter. This factor is composed of TBP (TATA binding protein) and other RNAP I-associated subunits. The yeast Schizosaccharomyces pombe is unusual in that the rRNA gene promoter contains a canonical TATAAA element upstream from the transcription initiation site. This TATA element, which normally binds the TBP subunit of RNA polymerase II initiation factor TFIID, plays an important role in directing the initiation of transcription of the S. pombe rRNA genes. This research will genetically and biochemically test the ability of the TBP subunit of S. pombe RNAP I essential transcription factor SL1 to direct assembly of the transcription pre-initiation complex via the TATAAA element. This analysis includes preparative purification of SL1, cloning and sequencing of at least one TBP-associated subunit, and identification of additional interacting factors by in vivo genetic analysis. The interaction of purified SL1 with a second apparently inhibitory promoter domain will be characterized. The vertebrate rDNA promoter/enhancer binding protein UBF (Upstream Binding Factor) also appears to play a pivotal role in stimulation and regulation of rRNA synthesis. An S. pombe UBF-like activity that specifically interacts with anti-UBF antiserum, and with both the rDNA promoter and intergenic enhancer region will be further characterized by purification of the UBF complex, cloning and sequencing of component subunits, and genetic and biochemical analysis of function. In addition, the DNA binding properties of another S. pombe intergenic rDNA binding protein, the RNAP I transcription termination factor Reb1p, will be defined with respect to binding affinities and sequence specificity. The S. pombe model system has potential application to a wide vari ety of eukaryotic organisms and general relevance to understanding the evolution of transcription. This research will analyze the molecular interactions governing activation and regulation of the large ribosomal RNA genes in eukaryotes. The yeast Schizosaccharomyces pombe has transcriptional mechanistic and regulatory features with potential application to a wide variety of eukaryotic organisms, and has general relevance to understanding the evolution of transcription in animals, plants and the more widely investigated yeast model system S. cerevisiae.
