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.

Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rm6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.

Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrngp, Rrnl0p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrnllp. UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex. The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases. We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF. Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element. By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element. We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrngp both in vitro and in the yeast two-hybrid system in vivo. Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter. Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF.[Key Words: Saccharomyces cerevisiae; TBP; UAF; CF; RNA polymerase I; rDNA promoter] Received July 15, 1996; revised version accepted August 21, 1996.In eukaryotes, each of the three nuclear RNA polymerases, RNA polymerase I (Pol I), RNA polymerase II (Pol II), and RNA polymerase III (Pol III), utilizes a different set of basic transcription factors for recognizing and binding to their respective promoters for initiation; polymerases themselves are unable to bind to promoters directly. The TATA-binding protein (TBP) is unique in that it is required for initiation of transcription by all three RNA polymerases (Hemandez 1993). TBP was originally identified as a protein component of TFIID that binds directly to the TATA box present in many Pol II promoters. For these promoters, the function of TBP is well established; TBP, either by itself or as a part of TFIID that also contains other TBP-associated proteins (TAFs), binds directly to the TATA box and recruits first TFIIA and TFIIB, which in turn recruits Pol II together with TFIIF and other basic transcription factors, such as 'Corresponding author.

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Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeastSaccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations in RRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations, L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.Transcription of ribosomal DNA (rDNA) by RNA polymerase I (Pol I) in Saccharomyces cerevisiae (referred to as yeast in this paper) utilizes at least four factors other than Pol I: upstream activation factor (UAF) (which includes Rrn5p, Rrn9p, Rrn10p, and at least two more proteins) (11), core factor (CF) (which contains Rrn6p, Rrn7p, and Rrn11p) (12, 15, 17), TATA-binding protein (TBP) (6,23,25), and Rrn3p (30). Like other eukaryotic rDNA promoters studied previously, the yeast rDNA promoter consists of two cis elements, the upstream element and the core promoter. The upstream element is required for a high level of transcription but is dispensable for a basal level of transcription, whereas the core promoter is essential for the accurate initiation of transcription (5,11,14,18). Our previous in vitro studies have demonstrated that UAF interacts with the upstream element of the rDNA promoter, forming a stable UAF-template complex and committing the template to transcription. UAF then recruits CF to the promoter, and TBP is required for this recruitment to form a stable preinitiation complex containing UAF, CF, and presumably TBP. Finally, with the aid of Rrn3p, Pol I joins this preinitiation complex, and the system becomes ready for transcription initiation (11,25,30).In agreement with the conclusion that UAF mediates the stimulatory function of the upstream element, UAF i...