Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes (ϳ143 in this case) and a strain in which the rRNA gene number was reduced to ϳ42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was ϳ50 ؎ 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 ؎ 29 polymerases/ gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of ϳ60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

No abstract

About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae. Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes. Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active. By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats. Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA.

In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify "ribosomal" Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer.