Nucleic Acids Research, 2010, Vol. 38, No. 16 5323 Figure 5. Reduction of Rrn3p-levels decreases rRNA production but does not phenocopy growth-inhibition and the loss of nascent rRNAs upon TOR inactivation. (A) Yeast strain pTet7-RRN3-Prot.A (TOY 667), expressing Rrn3p under the control of a doxycycline sensitive 7-fold TetO-promoter was grown in YPAD at 30 C to early log phase, before cells were split and further cultured in the absence or presence of 0.1, 0.5 and 1 mg/ml doxycycline (Dox), respectively. After 17 h, 50 ml of the cultures were collected and lysed and same amounts of WCE (30 mg) were analysed by western blotting, using an antibody directed against the Prot.A-tag of the Rrn3p. (B) Overexpression of Rrn3p results in more Pol I– Rrn3p complexes. Yeast strain pTet7-RRN3-Prot.A was grown in YPAD at 30 C without or in the presence of 1 mg/ml doxycycline to log-phase. After lysis, same amounts of WCE (900 mg) were separated on a Superose-6 column and further analysed as described in Figure 4A. Western blots were developed with anti-A135-antibodies which recognize at the same time also Rrn3p-Prot.A through its Prot.A-tag. Note that about equal amounts of Pol I in both strains were detected. The gel ﬁltration fractions in which Rrn3p co-migrates with Pol I are labelled in red. (C) Reduction of Rrn3p-levels decreases rRNA synthesis. Five millilitres aliquots of strain pTet7-RRN3-Prot.A growing in the presence of various doxycycline concentrations were pulse labelled for 15 min with 20 mCi of [5, 6-3H] uracil. Twenty percent of the isolated RNA were separated by denaturing gel electrophoresis and blotted onto a nylon membrane. 3H-labelled rRNAs were visualized using the BAS 1000 imaging system and quantiﬁed with the Image Gauge software. The graph on the bottom represents the 35S/27S nascent rRNA ratio determined in two separate experiments. The ratio in the 0 mg/ml Dox sample was arbitrarily set to 100. (D) Reduction of Rrn3p-levels upon TOR inactivation does not explain the complete shut oﬀ of pre-rRNA synthesis. Yeast strain RRN3-Prot.A (WT) expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30 C to early log phase (t = 0 min). One third of the culture was withdrawn before rapamycin was added to a ﬁnal concentration of 200 ng/ml and incubation was continued for 20 and 120 min, respectively (t = 20, 120 min). Yeast strain pTet7-RRN3-Prot.A (TOY 667) was grown in YPD in the presence of 0.2 mg/ml doxycycline (Dox) at 30 C for 18 h to early log phase, and treated with rapamycin as described above. From all samples cells from 50 ml were sedimentated and WCEs were prepared, from which 30 mg of protein were analysed by western blotting, using an antibody directed against the Prot.A-tag of the Rrn3p. In parallel, 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 RNAs were separated by denaturing gel electrophoresis and visualized by Ethidium bromide staining (EtBr). After transfer onto a nylon membrane 3H-labelled rRNAs were visualized as described in the legend to Figure 5C. (E) Chromatin-IP (ChIP) experiments. Yeast strain pTet7-RRN3-Prot.A (TOY 667) was grown in YPD at 30 C to mid-log phase in the presence of the indicated doxycycline concentrations. Cells were crosslinked and analysed as described for Figure 4C. (F) Rrn3p expression levels do not inﬂuence kinetics of growth inhibition upon TOR inactivation. Strain pTet7-RRN3-Prot.A was grown without or in the presence of 1 mg/ml doxycycline (DOX) and compared to strain RRN3-Prot.A (note that this is not the exact isogenic wild-type strain to pTet7-RRN3-Prot.A). The culture was diluted to an OD600 of 0.1. After 6 h half of the culture was withdrawn and rapamycin was added to a ﬁnal concentration of 200 ng/ml (indicated by an arrow). The OD600 of the culture was monitored hourly over the entire 10-h time-course of the experiment. 35S, 27S and 20S pre-rRNAs are equally diminished after doxycycline dependent Rrn3p-reduction. This is reﬂected in a constant quotient of newly synthesized 35S/27S rRNA (Figure 5C). Since processing defects generally lead to the distortion of the precursor rRNA ratios, we conclude that in this experiment Pol I transcription, but not rRNA processing is aﬀected. We further investigated the association of the Pol I subunit A43 with the promoter, 18S and 25S region of the rDNA in ChIP ex- periments in dependency of Rrn3p expression (Figure 5E). Doxycycline-dependent reduction of Rrn3p levels correlated well with a decrease in rDNA-associated Pol I. Taken together, our data demonstrate a correlation between Rrn3p levels, the formation of Rrn3p–Pol I