Nucleic Acids Research, 2010, Vol. 38, No. 16 5321 Figure 4. Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. (A) Gelﬁltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N) were grown in YPD at 30 C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 mg) were separated on a Superose-6 column in a buﬀer containing 1.5 M potassium acetate. An amount of 250 ml of the collected 500 ml fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 mg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel ﬁltration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. (B) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 ( N), both expressing chromosomally HA3-tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30 C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed (t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above (t = 120 min). The HA3-tagged Pol I subunit A43 was immunoprecipitated (a-HA-IPs) from 250 ml of extracts (Inputs) with anti-HA antibody. Fifty percent of the a-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3- N-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantiﬁed using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. (C) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N), both expressing either chromosomally HA3-tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30 C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and soniﬁed. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA3-tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of speciﬁc DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers speciﬁc for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. (D) Reduction of 35S pre-rRNA synthesis is attenuated in the N-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N) were cultured to mid-log phase (t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 mCi of [5, 6-3H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3H-labelled RNAs were visualized and quantiﬁed using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantiﬁed as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the diﬀerent rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100. signiﬁcantly higher upon TOR inactivation (Figure 4B, compare lanes 9 and 10 with lanes 7 and 8). This result indicates that in nutrient-depleted wild-type cells endogen- ous Rrn3p-levels and (as a consequence) the number of Rrn3p–Pol I complexes are reduced. On the other hand, some reduction of Rrn3p–Pol I complexes is also observed if TOR is inhibited in the N-mutant. In the experiment depicted in Figure 4B, 64% N-Rrn3p-Prot.A remained bound to Pol I (Figure 4B, lanes 9 and 10), although the Rrn3p levels are very similar before and after nutrient starvation (Figure 4B, lanes 3 and 4). Similar results were obtained when Rrn3p–Pol I complexes were analysed by co-immunoprecipitation in the cim3-1 mutant (Supplementary Figure S3B). Accordingly, the re- duction of endogenous Rrn3p levels is one important cellular process to decrease the amount of Pol I–Rrn3p complexes, but there are additional mechanisms, which contribute.