Alternative Chromatin Structures of the 35S rRNA Genes in Saccharomyces cerevisiae Provide a Molecular Basis for the Selective Recruitment of RNA Polymerases I and II

Abstract

In all eukaryotes, a specialized enzyme, RNA polymerase I (Pol I), is dedicated to transcribe the 35S rRNA gene from a multicopy gene cluster, the ribosomal DNA (rDNA). In certain Saccharomyces cerevisiae mutants, 35S rRNA genes can be transcribed by RNA polymerase II (Pol II). In these mutants, rDNA silencing of Pol II transcription is impaired. It has been speculated that upstream activating factor (UAF), which binds to a specific DNA element within the Pol I promoter, plays a crucial role in forming chromatin structures responsible for polymerase specificity and silencing at the rDNA locus. We therefore performed an in-depth analysis of chromatin structure and composition in different mutant backgrounds. We demonstrate that chromatin architecture of the entire Pol I-transcribed region is substantially altered in the absence of UAF, allowing RNA polymerases II and III to access DNA elements flanking a Pol promoter-proximal Reb1 binding site. Furthermore, lack of UAF leads to the loss of Sir2 from rDNA, correlating with impaired Pol II silencing. This analysis of rDNA chromatin provides a molecular basis, explaining many phenotypes observed in previous genetic analyses.

Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae

“…In contrast to the results obtained at the PHO5 locus, the MNase fusion proteins cleaved to a similar extent within the IGS region, regardless if PHO5 expression had been activated or repressed (Figure 7B compare lanes 1–4, 9–12, and 17–20 with lanes 5–8, 13–16, and 21–24, respectively). As observed earlier (35), TBP/Spt15-MNase dependent cleavage events could be observed at the Pol I promoter, the Pol III promoter, E-Pro and the ARS region (Figure 7B, lanes 1–8), consistent with the results of the western blot analysis (Figure 2B). Rpb3-MNase and Snf6-MNase-mediated cleavage was mainly restricted to the E-pro region, with additional weak cleavage events of Rbp3-MN at the ARS and at a site directly upstream of the Pol I promoter (Figure 7B, lanes 9–16 and 17–24).…”

Reduction in Ribosomal Protein Synthesis Is Sufficient To Explain Major Effects on Ribosome Production after Short-Term TOR Inactivation in Saccharomyces cerevisiae

“…Hmo1-MNase produced a very similar cleavage pattern ( Fig. 2A, lanes 13 to 15 and 18 to 20), consistent with earlier results (22,52). Consistent with the result of Rpa43-HA ChIP experiments, we did not observe any qualitative or quantitative changes in cleavage by the MNase fusion proteins upon rapamycin treatment for 15 min (Fig.…”

“…Chromatin immunoprecipitation (ChIP) was essentially performed as described elsewhere (22). Primer pairs used for amplification were 969 (5Ј-TCA TGG AGT ACA AGT GTG AGG A-3Ј) and 970 (5Ј-TAA CGA ACG ACA AGC CTA CTC-3Ј), 712 (5Ј-GAG TCC TTG TGG CTC TTG GC-3Ј) and 713 (5Ј-AAT ACT GAT GCC CCC GAC C-3Ј), 710 (5Ј-TGG AGC AAA GAA ATC ACC GC-3Ј) and 711 (5Ј-CCG CTG GAT TAT GGC TGA AC-3Ј), and 920 (5Ј-GCC ATA TCT ACC AGA AAG CAC C-3Ј) and 921 (5Ј-GAT TGC AGC ACC TGA GTT TCG-3Ј).…”

TFIIB-related factors in RNA polymerase I transcription

“…In vivo, chromatin immunoprecipitation (ChIP) studies show that Pol I crosslinks to the promoter and rDNA coding regions as expected, whereas UAF, CF, and Rrn3 are exclusively promoter associated [41]. In the absence of Pol I and Rrn3, UAF, CF, and TBP remain stably bound to the promoter, suggesting that CF and TBP build a reinitiation scaffold in vivo [42]. …”