It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

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.

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30°C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.yeast Saccharomyces cerevisiae T he synthesis of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I is an important step in the synthesis of ribosomes, and its regulation is closely linked to the nutrient conditions and growth potential for the cell. Previous studies have identified essential components of the Pol I transcription apparatus as well as important cis-elements in Pol I promoters and revealed some potential mechanisms for regulation of transcription initiation (for reviews, see refs. 1-5). Unlike Pol II, however, little is known regarding mechanisms that regulate postinitiation steps of transcription by Pol I.One well characterized Pol II transcription elongation factor in yeast is a complex of two proteins, Spt4p and Spt5p (the complex will be referred to as Spt4͞5 here). The SPT4 and SPT5 genes were among many genes isolated for their ability to suppress transcription defects caused by insertions of the retrotransposon Ty1 (or the Ty1 long terminal repeats, ␦) in the 5Ј noncoding regions of yeast genes (6). Swanson and Winston (7) later showed that Spt4͞5 associates with Spt6p, which affects Pol II elongation through chromatin. However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4͞5 in transcription elongation by Pol II in yeast.Spt4͞5 also plays a role in Pol II transcription elongation in mammalian cells. The mammalian homologues of Spt4p and Spt5p form a complex called the 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which was originally identified as a factor that induces a DRB-dependent arrest of elongating Pol II complexes in reconstituted in vitro transcription assays (11). It was demonstrated that under nucleotide-limiting conditions, DSIF could also increase the rate of elongation of Pol II in vitro. Thus, work in mam...

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.