Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex.

Abstract

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.

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

“…First, it is formed by several hundreds of tandem repeats of about 10 kb in yeast that cluster together to form the nucleolus [1]. Second, promoters contain two main regions termed upstream element (UE) and core element (CE) in yeast, with the latter overlapping with the transcription start site and the former extending to about 150 bp upstream of this site [2]. Third, in actively growing cells rDNA transcription represents 60% of the total transcriptional activity [3], which correlates with a high content of RNA polymerase occupancy on rDNA genes [4].…”

“…The upstream activating factor (UAF) binds the UE [2], while the core factor (CF) binds the CE [8]. The two protein complexes interact with each other and are bridged through TATA-box binding protein (TBP) binding [9].…”

Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants

“…In the case of 64% deletion, all four spores were viable and formed colonies after 4 days of incubation at 30uC; however, the disruption of 80% of ORF YLR141w yielded only two viable spores, indicating that YLR141w is an essential gene. In contrast, Keys et al (1996) reported that RRN5 deletion is not lethal because all spores from the rrn5D/RRN5 diploid were capable of forming colonies. The spores containing the disruption normally formed two microcolonies only after 10 days of incubation, although sometimes only one or neither of the spores grew.…”

“…Tetrad analysis showed that such deletions were not lethal and produced no apparent phenotype in the haploid cell. Keys et al (1996) reported that YLR141w is the RRN5 gene, an RNA polymerase I-specific transcription factor, whose function is not essential but is required for high level of transcription. This prompted a further deletion of 200 bp more of the upstream region of ORF YLR141w, resulting in a total of 78% disruption.…”

“…Since the work was initiated, the function of four ORFs has been assigned. YLR141w was identified as the RRN5 gene that codes for one member of the multiprotein transcription factor, UAF (upstream activation factor), required for high level of transcription of rDNA (Keys et al, 1996). YLR144c is the assembly complementing factor (ACF2), a component of cortical actin assembly sites in polarized yeast cells that is involved in cell surface growth and shape definition (Lechler and Li, 1997).…”