5324 Nucleic Acids Research, 2010, Vol. 38, No. 16 complexes, recruitment of Pol I to the rRNA genes, rRNA gene transcription and the growth rate in yeast. In the presence of 0.2 mg/ml doxycycline the Rrn3p level in pTet7-RRN3-Prot.A cells was very similar to the Rrn3p level in wild-type cells after 2 h of rapamycin treatment (Figure 5D, upper panel, compare lanes 3 and 4). Strikingly, we observed large diﬀerences in the pattern of newly synthesized (pre)-rRNA in these two conditions: As noted before (Figure 5C), doxycyline treatment aﬀected the diﬀerent precursors similarly, with no evidence for defective processing (Figure 5D, compare lane 4 with lane 1). After 20 min of rapamycin treatment Rrn3p levels did not drop signiﬁcantly, but rRNA labelling and processing were strongly aﬀected. [Note, that cellular uptake of uracil was only modestly aﬀected after 20 min rapamycin treatment (data not shown, and Reiter et al., submitted)]. Accordingly, substantial amounts of newly synthesized 35S pre-rRNA are still detected, whereas label incorporation in matured rRNA species was reduced in rapamycin treated cells [compare Figure 5D, lane 4 with lane 2/3; lane 1 with lane 2/3, and lane 4 with lane 5/6; see also (45)]. Furthermore, in contrast to the doxycycline-treated pTet7-RRN3-Prot.A strain, rapamycin treated cultures stopped growth shortly after addition of the drug (Figure 5F). Rapamycin dependent growth arrest occurred with the same kinetics [about 1 h after addition of rapamycin (arrow in Figure 5F)], no matter whether Rrn3p was overexpressed or scarce (Figure 5F). Therefore, the simple reduction of Rrn3p-levels and con- sequently the decreased association of the Pol I-machinery with rDNA do not fully explain the dramatic eﬀects on ribosome neo-production and cellular growth upon TOR inhibition. DISCUSSION Our analyses conﬁrm that in yeast the complex control of ribosome biosynthesis occurs on several layers. One of them is the proteasome-dependent degradation of the transcription factor Rrn3p which contributes to the down-regulation of transcription initiation as a conse- quence of TOR inactivation by rapamycin. Several obser- vations indicate that lowered cellular Rrn3p levels after TOR inactivation are not the result of a targeted destruc- tion [see as review (47)], but rather the combination of a reduced RRN3 expression rate and a constitutive short half life of the protein (48): ﬁrst, RRN3 mRNA levels quickly decrease upon nutrient depletion. [(38,49) and our results, not shown]. Second, both inactivation of Pol II-dependent transcription of the RRN3 gene in Pol II conditional mutant and inhibition of RRN3 mRNA trans- lation by cycloheximide reduce Rrn3p protein levels with similar kinetics as TOR inactivation, thus, TOR inactiva- tion aﬀects endogenous Rrn3p levels very likely at the stage of or prior to protein production. Ubiquitylation of Rrn3p seems to be an evolutionary conserved feature since this modiﬁcation has recently been reported for the mammalian homologue, TIF-IA (50). However, in contrast to the endogenous yeast Rrn3p, TOR-inactivation does not lead to reduced levels of ectopically co-expressed TIF-IA in the mammalian system (12). Thus, it appears that—although human TIF-IA/Rrn3p substitutes the yeast homologue for growth (51)—their activities may be diﬀerently regulated. It is conceivable that the requirements to respond to incoming signals diﬀer between a rapidly dividing yeast cell and higher eukaryotic cells which have more time to adjust to environmental situations. Nomura and co-workers (11,40) reported that the TOR pathway regulates Rrn3p-dependent recruitment of Pol I to the rDNA. Using ChIP assays and EM Miller spreading technology they calculated that inhibition of the TOR pathway reduces the Pol I-coverage on the rDNA gene to about 40–50% of the normal levels (40) . In addition, they observed that the amount of Pol I associated with Rrn3p was reduced by a factor of about three after TOR inactivation by rapamycin (11). Our data suggest that decreasing cellular Rrn3p levels after TOR inactivation is one important cellular mechanism to reduce the amount of Pol I–Rrn3p complexes. On the other hand, in cells expressing a stabilized Rrn3p variant we still observed some reduction in the level of Pol I– Rrn3p complexes after nutrient depletion indicating that Rrn3p association with Pol I can also be regulated by other means. The kinase Tor1p was reported to be associated with the rDNA promoter in exponentially growing cells, whereas it shuttles to the cytoplasm upon TOR inactivation (52). Thus, it is possible that nuclear Tor1p directly modulates the formation and dissociation of the Pol I–Rrn3p complex. Experiments using a non-dissociable Pol I–Rrn3p fusion protein (CARA for Constitutive Association of Rrn3 and A43) are in general agreement with our conclusions (25). The CARA strain mimics an active non-dissociable Pol I– Rrn3p complex, which is resistant to rapamycin treatment. Similar to the N mutant analysed in this study this strain shows an attenuation of the rapamycin-dependent repres- sion of transcription. However, the retention of Pol I at the rDNA upon rapamycin treatment is signiﬁcantly higher in the CARA strain than in the N mutant strain upon nutrient starvation [compare Figure 4C in this study with Figure 2B in (25)]. This is consistent with the interpret- ation, that in the CARA strain, both, the decrease in Rrn3p protein levels, and the Rrn3p-A43 dissociation are prevented. On the other hand, also in CARA expressing cells overall rRNA synthesis is substantially reduced upon rapamycin treatment (Figure 4D in (25)). This indicates the existence of other mechanisms regulating rRNA produc- tion independent of the formation of the Pol I–Rrn3p complex also in this experimental system. If we adjust Rrn3p levels to values substantially below the reduced amounts of Rrn3p observed upon TOR in- activation by rapamycin, we ﬁnd a signiﬁcant reduction in rRNA synthesis. In contrast to TOR inactivation, however, mature rRNAs are still produced even if amounts of initiation competent Pol I–Rrn3p complexes are minimal. We conclude that nutrient dependent regu- lation of cellular Rrn3p levels alters rDNA transcription on the level of the (re-) initiation rate (11,42) but does not fully account for the TOR-dependent shut-down of