Components of the SSU-processome colocalize at the 5′-end of the 25S gene. (A) Yeast rDNA unit. The Pol I transcript extends from the promoter (P) to the terminator (T) regions of the rDNA and encodes the 18S, 5.8S, and 25S rRNAs. Mature rRNAs are interspersed with external (5′- and 3′-ETS) and internal (ITS1 and ITS2) transcribed spacers. 5S is transcribed by Pol III in the opposite direction. 5S is embedded in nontranscribed regions 1 and 2 (NTS1 and NTS2). Amplicons used in the Ch-IP analysis are indicated (1, 3, 6, 5′-ETS, 7, 9–14, and 18). Scale bar is 500 bp. (B) Distribution of the largest subunit of Pol I (Rpa190) along the rDNA unit. Chromatin extracts prepared from yeast cells expressing either a functional carboxyl-terminal HA or TAP epitope tagged version of Rpa190, and otherwise isogenic wild-type strains (TAP and HA), grown to mid-log phase were submitted to coprecipitation analysis with IgG-coated magnetic beads and the copurifying DNAs specifically analyzed by qPCR with oligonucleotide pairs specific to the entire stretch of the rDNA locus (A). Fold enrichment at each position (color-coded as in panel A) corresponds to the IP/input ratio normalized to amplicon 3. The threshold of significant enrichment was arbitrarily set to 2 (dashed line). (C,F) Distribution along the rDNA locus of representative UTP proteins from the UTP-A, UTP-B, and UTP-C subcomplexes of the SSU-processome (Utp1, Utp9, and Utp22), as well as that of unrelated Utp7, expressed either as functional carboxyl-terminal TAP (panel C) or GFP (panel F) epitope tagged constructs. (D) Distribution along the rDNA locus of 6 Utps from the UTP-A subcomplex. (E) Distribution of Pol5 (a suspected UTP-A member), Nop15 (a nucleolar protein involved in 60S ribosome synthesis), and Ent5 (a cytoplasmic protein) along the rDNA locus. (C–F, upper panels) Ch-IP analysis in yeast strains expressing functional carboxyl-terminal TAP- (panels C–E) or GFP (panel F)-tagged fusion of each bait protein. (Lower panels) Western blot analysis of protein coprecipitation efficiency. Chromatin extracts from the total (T), supernatant (S), and pellet (P) fractions were loaded in a 1:1:10 ratio in SDS-PAGE and analyzed by quantitative Western blot (ECLplex, panels C–E) or regular ECL (panel F). In the ECLplex analysis, the value obtained for the T fraction was set to 100 and the IP efficiency expressed as P/T × 100 ratio.
