5316 Nucleic Acids Research, 2010, Vol. 38, No. 16 Although regulation of rRNA synthesis is an important TOR function and several targets of TOR signalling af- fecting the Pol I-machinery were proposed, TOR controls ribosome biogenesis also by stimulating transcription of ribosomal protein genes (19–21) and mRNA translation, the latter especially through translation initiation factor 4E-binding proteins and through the S6 kinase (S6K) [see as review (22)]. Whether and how TOR controls these diﬀerent processes in a coordinated manner is not understood. In Saccharomyces cerevisiae, formation of initiation complexes at the 35S ribosomal DNA (hereafter called rDNA, rRNA gene) promoter depends on the cooperate activity of upstream activating factor (UAF) with TATA-binding protein, core factor (CF) and initiation competent Pol I which is tightly associated with Rrn3p [reviewed in (3,23)]. Availability of the Pol I–Rrn3p complex represents a limiting step to support 35S rRNA gene transcription. In cells grown to stationary phase or treated with rapamycin, which impairs TOR signalling, the amounts of Pol I–Rrn3p complexes were signiﬁcantly reduced (11,24). In these conditions Rrn3p-dependent re- cruitment of Pol I to the rDNA promoter is impaired (11). Concordantly, in yeast cells expressing a covalently bound, non-dissociable complex between Rrn3p and Pol I, down-regulation of rDNA transcription is delayed and Pol I occupancy at the rDNA promoter remains high after inactivation of the TOR pathway (25). We investigated how nutrient deprivation aﬀects rDNA transcription in yeast and found that Rrn3p-levels are rapidly reduced upon TOR inactivation whereas Pol I-levels remain unaﬀected. Rrn3p is constitutively degraded by the proteasome and Rrn3p levels decrease as soon as synthesis of the protein is derogated. We found that degradation of Rrn3p correlates with the loss of Pol I–Rrn3p complexes and a reduced Pol I occupancy at the 35S rDNA promoter and coding region. To analyse if Rrn3p degradation plays a major role in the down-regulation of rRNA gene transcription in starved cells, we analysed mutant strains in which Rrn3p levels are unaﬀected upon TOR inactivation. We found that in these cells Pol I–Rrn3p complex formation and Pol I recruitment to rDNA is only marginally impaired by TOR inactivation while growth inhibition and shut-down of the synthesis of mature rRNAs cannot be rescued. By using the Tet-oﬀ system to adjust deﬁned Rrn3p levels we demonstrate that decreasing the amount of cellular Rrn3p results in reduced rDNA transcription and growth rate. However, even minimal amounts of Rrn3p support rRNA production and cell growth. We conclude that the almost complete inhibition of ribosome synthesis upon TOR inactivation cannot fully be explained by Rrn3p degradation and thus reduced rDNA transcription initiation. MATERIALS AND METHODS Yeast strains, plasmids and media See Supplementary Data. Preparation of yeast whole cell extracts Yeast cells were harvested by centrifugation (3 min, 3000g, 4 C) and washed once with cold H2OMillipore. After sus- pending the pellets in the same volume of cold lysis buﬀer [20% (w/v) glycerol, 150 mM HEPES (pH 7.6), 10 mM MgCl2, 400 mM (NH4)2SO4, 5 mM b-mercaptoethanol, 1 mM PMSF, 2 mM benzamidine], cells were broken for 45 min with pre-cooled glass beads (Ø 0.75–1.0 mm, Roth) on a VXR basic Vibrax (IKA) with 2000–2200 r.p.m. at 4 C. The suspensions were mixed with 150–250 ml of cold lysis buﬀer and glass beads and cell debris were sedi- mented (15 min, 13 000 r.p.m., 4 C). The supernatants [whole cell extracts (WCEs)] were frozen in liquid nitrogen and stored at 80 C. Isolation of (poly)ubiquitylated proteins from yeast extracts WCEs were adjusted to 750 mM KOAc, 0.5% (v/v) Nonidet P40, 0.05% (v/v) Triton X-100 and incubated with recombinant GST-Dsk2p bound to 50 ml glutathione sepharose (GE Healthcare). Dsk2p is an ubiquitin-binding protein. A mock isolation using recombinant GST bound to 50 ml glutathione sepharose (GE Healthcare) served as a control. After batch binding beads were washed twice with 750 mM KOAc, 0.5% Nonidet P40 and twice with PBS. Precipitated proteins were eluted from the beads with SDS sample buﬀer and analysed by western blotting. In vivo pulse labelling and northern blot experiments After harvesting 3–5 ml of yeast cultures, cells were sus- pended in 1 ml of the respective medium and pulse-labelled for 15 min with 20 mCi [5, 6-3H] uracil (Amersham) at 30 C. Total RNA was isolated by hot-phenol extraction and ethanol-sodium acetate precipi- tation (26), separated in a denaturing 1.3% agarose gel and transferred onto a nylon membrane (PositiveTM, Qbiogene). 3H-labelled rRNAs were visualized using a BAS-MS 2040 imaging plate (Fujiﬁlm) and a BAS 1000 phosphorimager (Fujiﬁlm, 4–5 days exposition). Quantiﬁcation was performed using the Image Gauge software (Fujiﬁlm). For northern blot analysis membranes were hybridized with a 32P-labelled 25S oligonucleotide probe (#212: 50-CTC CGC TTA TTG ATA TGC-30) using the RadPrime DNA labelling system (Invitrogen) with incorp- oration of [a-32P]dATP (Hartmann analytic) according to the instructions of the manufacturer. Quantiﬁcation was performed as described above, but using a BAS-III imaging plate (Fujiﬁlm). Gelﬁltration of yeast WCEs Yeast WCEs were ﬁrst clariﬁed by centrifugation (40 min, 100 000g, 4 C). The supernatants (900 mg of protein) were loaded on a Superose 6 HR 10/30 column (FPLC , Pharmacia Biotech) and separated in a high salt buﬀer [10% (w/v) glycerol, 20 mM HEPES (pH 7.8), 2 mM MgCl2, 0.02 mM EDTA, 1.5 M potassium acetate, 0.05% (v/v) Tween 20, 5 mM b-mercaptoethanol, 1 mM PMSF, 2 mM benzamidine] at a ﬂow rate of