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Keys, DA, Lee, BS, Dodd, JA, Nguyen, TT, Vu, L, Fantino, E, Burson, LM, Nogi, Y and Nomura, M (1996) Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex. Genes Dev. 10:887-903
Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.
Amino Acid Sequence; Base Sequence; Cell-Free System; Cloning, Molecular; Crosses, Genetic; DNA, Ribosomal; DNA-Binding Proteins/isolation & purification; DNA-Binding Proteins/metabolism; Genes, Fungal; Genetic Complementation Test; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; RNA Polymerase I/metabolism; RNA, Ribosomal/biosynthesis; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA; Sequence Deletion; Templates, Genetic; Transcription Factors/isolation & purification; Transcription Factors/metabolism; Transcription, Genetic