5322 Nucleic Acids Research, 2010, Vol. 38, No. 16 Preserving the number of Pol I–Rrn3p complexes attenuates the decrease of Rrn3p and Pol I association with rRNA genes and the shut down of transcription upon nutrient depletion To investigate whether stable levels of Pol I–Rrn3p complexes in the N-mutant correlate with robust recruit- ment of Pol I to the rDNA even after inactivation of the TOR pathway, we performed ChIP (chromatin immunoprecipitation) experiments. In good agreement with previous results where TOR signalling was inhibited by the addition of rapamycin (11,40), we observe a decrease of Pol I promoter DNA and DNA fragments of the 35S rRNA coding sequence co-precipitating with Pol I in extracts from wild-type cells being starved of es- sential amino acids to about 40% of the co-precipitation before starvation (Figure 4C, central panel). The same was observed for Pol I promoter DNA co-purifying with Rrn3p (Figure 4C, left panel). In contrast, in extracts of the N-mutant co-precipitation of the respective rDNA fragments with either Rrn3p or Pol I decreased only to about 60% upon starvation (Figure 4C). Accordingly, the reduction in endogenous Rrn3p levels when nutrients become limiting signiﬁcantly contributes to but does not fully explain the decreased Pol I loading on rRNA genes in this situation. Nutrient depletion did not lead to signiﬁ- cant changes in the association of promoter bound subunits of the transcription factor CF (Figure 4C, right panel) and UAF [data not shown, see also (11)] in wild-type cells and the N-mutant. Thus, CF and UAF remain stably associated with the rDNA promoter upon TOR inactivation although the formation and recruitment of the Pol I–Rrn3p complex is impaired. Interestingly, the absolute amount of rDNA co-precipitating with Pol I, Rrn3p and CF was modestly increased in the N-mutant. Since only half out of the 150 rRNA genes of the yeast rDNA locus are actively transcribed in wild-type conditions, this could mean that this number is slightly enhanced in strains carrying the mutant rrn3 allele. Electronmicroscopic inspection of rRNA genes by the Miller chromatin spreading method (41,42) suggested a higher density of actively transcribed rDNA genes in individual nucleolar regions released from N-mutant cells when compared to nucleolar regions released from wild-type cells (Supplementary Figure S4A). To validate the impression obtained in these single-cell analyses, we turned to psoralen crosslinking ex- periments in which actively transcribed and transcription- al inactive rRNA genes can be distinguished and are averaged over a large cell population (30,43,44). We observed a slight increase (<15%) in the number of actively transcribed rRNA genes in the N-mutant in direct comparison with the corresponding wild-type strain (Supplementary Figure S4B) providing a possible explanation for the increased amount of 35S rRNA gene segments co-precipitating with Rrn3p and Pol I in the mutant strain compared to the corresponding wild-type strain in the above ChIP experiments (Figure 4C). To investigate how stabilization of Rrn3p levels upon TOR inactivation inﬂuences rRNA-production we examined new synthesis of the primary rRNA transcript in the wild-type strain and the N-mutant. We performed pulse labelling experiments with 3H uracil and found that in the wild-type strain, a signiﬁcant amount of 35S rRNA precursor is still produced after 30 min and even— although to a lower extent—after 60 min of rapamycin treatment (Figure 4D). In the mutant strain expressing Rrn3p- N-Prot.A the ratio of newly synthesized 35S rRNA to steady state 25S rRNA levels is higher at each time-point (up to 2-fold after 30 min of TOR inactivation), when compared to the 35S/25S rRNA ratio in a wild-type strain (Figure 4D, graph on the right) arguing again for a partial deregulation of rDNA transcription in the N-mutant when TOR is inactivated. Taken together these data indicate that (i) after TOR inactivation rDNA transcription is reduced, but still occurs after up to 60 min of rapamycin treatment [see also (11,45)] and (ii) the re- duction of endogenous Rrn3p levels under those condi- tions contributes to the down-regulation of rDNA transcription. Lowered Rrn3p levels aﬀect Pol I–Rrn3p complex formation, 35S pre-rRNA synthesis and cellular growth, but do not fully mimic the response to TOR inactivation To uncouple eﬀects of an altered Rrn3p level from other eﬀects mediated by TOR inactivation, we generated a strain in which the only copy of the RRN3 gene was ex- pressed from a plasmid under the control of a doxycycline repressible 7-fold TetO promoter (pTet7-RRN3-Prot.A) (46). Using distinct amounts of doxycycline the expression levels of Rrn3p could be well controlled resulting in a strong down-regulation in the presence of 1 mg/ml doxy- cycline and a strong overexpression in the absence of the drug (Figure 5A, lanes 1 and 4). Interestingly, the strong decrease in Rrn3p level observed in the presence of 1 mg/ml doxycycline retarded, but did not completely inhibit growth (Figure 5E). Gelﬁltration of whole cell extracts derived from strains in which Rrn3p expression was either enhanced or reduced demonstrated again that Rrn3p levels correlated well with the number of Pol I–Rrn3p complexes (Figure 5B). Thus, changes in the level of Rrn3p did not signiﬁcantly alter the elution proﬁle of the protein in gelﬁltration. This means that a similar ratio of Rrn3p within a given population is monomeric, associated with Pol I, or part of a higher molecular weight complex (compare Supplementary Figure S3A with Figure 4A and Figure 5B). Apparently, there is a cellular mechanism maintaining a ﬁxed ratio between free and complexed Rrn3p. Next, we wanted to see how rRNA synthesis correlates with pTet7-controlled Rrn3p expression-levels. pTet7- RRN3-Prot.A cells were cultured with diﬀerent amounts of doxycycline and were pulse labelled for 15 min with 3H uracil to monitor rRNA synthesis. We found a signiﬁcant reduction of newly synthesized rRNA with increasing doxycycline concentrations (Figure 5C). The amount of nascent rRNA levels detected in pulse labelling experiments depends on the one hand on the synthesis of (pre-)rRNA due to Pol I dependent transcription and/or on the other hand, on their turnover due to deg- radation or productive processing events. Amounts of